U.S. patent number 10,914,747 [Application Number 15/769,237] was granted by the patent office on 2021-02-09 for immunoassay to detect cleaved high molecular weight kininogen.
This patent grant is currently assigned to Dyax Corp.. The grantee listed for this patent is Dyax Corp.. Invention is credited to Janja Cosic, Ryan Faucette, Daniel J. Sexton.
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United States Patent |
10,914,747 |
Sexton , et al. |
February 9, 2021 |
Immunoassay to detect cleaved high molecular weight kininogen
Abstract
The present disclosure provides immunoassay methods of detecting
a cleaved high molecular weight kininogen (HMWK) with high
sensitivity and specificity and isolated antibodies that
specifically bind cleaved HMWK.
Inventors: |
Sexton; Daniel J. (Melrose,
MA), Faucette; Ryan (Melrose, MA), Cosic; Janja
(Arlington, MA) |
Applicant: |
Name |
City |
State |
Country |
Type |
Dyax Corp. |
Lexington |
MA |
US |
|
|
Assignee: |
Dyax Corp. (Lexington,
MA)
|
Family
ID: |
1000005351067 |
Appl.
No.: |
15/769,237 |
Filed: |
October 19, 2016 |
PCT
Filed: |
October 19, 2016 |
PCT No.: |
PCT/US2016/057640 |
371(c)(1),(2),(4) Date: |
April 18, 2018 |
PCT
Pub. No.: |
WO2017/070170 |
PCT
Pub. Date: |
April 27, 2017 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20180306807 A1 |
Oct 25, 2018 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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62335311 |
May 12, 2016 |
|
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62243505 |
Oct 19, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N
33/54306 (20130101); C07K 16/36 (20130101); G01N
33/86 (20130101); G01N 33/6893 (20130101); G01N
2800/50 (20130101); G01N 2333/745 (20130101) |
Current International
Class: |
G01N
33/86 (20060101); G01N 33/543 (20060101); G01N
33/68 (20060101); C07K 16/36 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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102405228 |
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Apr 2012 |
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CN |
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102762203 |
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Oct 2012 |
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CN |
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0 210 029 |
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Jan 1987 |
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EP |
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S63-185398 |
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Jul 1988 |
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JP |
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WO 2006/101387 |
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Sep 2006 |
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WO |
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WO 2007/079096 |
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Jul 2007 |
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WO |
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WO 2011/075684 |
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Jun 2011 |
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WO |
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WO 2012/094587 |
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Jul 2012 |
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WO |
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WO 2012/170945 |
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Dec 2012 |
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WO |
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WO 2012/170947 |
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Dec 2012 |
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WO |
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WO 2014/113712 |
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Jul 2014 |
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WO |
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WO 2015/061182 |
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Apr 2015 |
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WO |
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WO 2015/061183 |
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Apr 2015 |
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WO |
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Primary Examiner: Gabel; Gailene
Attorney, Agent or Firm: Wolf, Greenfield & Sacks,
P.C.
Parent Case Text
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a national stage filing under 35 U.S.C. 371 of
International Patent Application Serial No. PCT/US2016/057640,
filed Oct. 19, 2016, entitled "IMMUNOASSAY TO DETECT CLEAVED HIGH
MOLECULAR WEIGHT KININOGEN", which claims the benefit of U.S.
Provisional Application Ser. No. 62/243,505, filed Oct. 19, 2015,
and 62/335,311, filed May 12, 2016 under 35 U.S.C. .sctn. 119, the
entire content of each of which is herein incorporated by
reference.
Claims
What is claimed is:
1. An immunoassay for determining a level of a cleaved high
molecular weight kininogen (HMWK), the immunoassay comprising: (i)
providing a support member, on which a first agent that
specifically binds a cleaved HMWK is immobilized; (ii) contacting
the support member of (i) with a biological sample suspected of
containing the cleaved HMWK; (iii) contacting the support member
obtained in (ii) with a second agent that binds HMWK, wherein the
second agent is conjugated to a label; and (iv) detecting a signal
released from the label of the second agent that is bound to the
support member, to determine the level of the cleaved HMWK in the
biological sample, wherein the first agent is an antibody
comprising a heavy chain complementarity determining region (CDR) 1
sequence FSFYVMV, a heavy chain CDR2 sequence GISPSGGNTAYADSVK, and
a heavy chain CDR3 sequence KLFYYDDTKGYFDF and a light chain CDR1
sequence SGSSSNIGSNYVY, a light chain CDR2 sequence RNNQRPS, and a
light chain CDR3 sequence AWDDSLNGRV.
2. The immunoassay of claim 1, wherein the support member is a
96-well plate.
3. The immunoassay of claim 1, wherein, prior to step (ii), the
support member of (i) is incubated with a blocking buffer.
4. The immunoassay of claim 1, wherein the second agent is a
polyclonal antibody, a monoclonal antibody, or a mixture of two or
more monoclonal antibodies that bind to HMWK.
5. The immunoassay of claim 1, wherein the label is a signal
releasing agent, wherein the signal releasing agent is a dye or
fluorophore.
6. The immunoassay of claim 1, wherein the label is a member of a
receptor-ligand pair and the immunoassay further comprises, prior
to step (iv), contacting the second agent in (iii) that is bound to
the support member, with the other member of the receptor-ligand
pair, wherein the other member is conjugated to a signal releasing
agent.
7. The immunoassay of claim 6, wherein the receptor-ligand pair is
biotin and streptavidin.
8. The immunoassay of claim 1, wherein the immunoassay is a Western
blot assay, an enzyme-linked immunosorbent assay (ELISA), or a
lateral flow assay.
9. The immunoassay of claim 1, wherein step (ii) is performed in
the presence of ZnCl.sub.1.
10. The immunoassay of claim 1, wherein the biological sample is
obtained from a human subject.
11. The immunoassay of claim 10, wherein the biological sample is a
serum sample or plasma sample, which is processed from a blood
sample collected in an evacuated blood collection tube comprising
one or more protease inhibitors.
12. The immunoassay of claim 10, wherein the human subject has a
disease and wherein the immunoassay further comprises determining
whether the disease is mediated by plasma kallikrein (pKal) based
on the level of the cleaved HMWK determined in step (iv), wherein
if the level of the cleaved HMWK is greater than a reference value,
the disease is mediated by pKal.
13. The immunoassay of claim 10, further comprising determining
whether the human subject has or is at risk for a disease mediated
by pKal, wherein if the level of the cleaved HMWK is greater than a
reference value, the subject is identified as having or at risk of
having the disease.
14. The immunoassay of claim 13, further comprising administering
to the subject an effective amount of a therapeutic agent for
treating the disease, if the subject is identified as having the
disease.
15. The immunoassay of claim 14, wherein the therapeutic agent is a
plasma kallikrein (pKal) inhibitor, a bradykinin 2 receptor (B2R)
inhibitor, and/or a C1 esterase inhibitor.
16. The immunoassay of claim 15, wherein the pKal inhibitor is an
anti-pKal antibody or an inhibitory peptide.
17. The immunoassay of claim 15, wherein the therapeutic agent is
lanadelumab, ecallantide, icatibant, or human plasma-derived C
1-INH.
18. The immunoassay of claim 10, wherein the human subject is on a
treatment for a disease mediated by pKal, and wherein the method
further comprises assessing the efficacy of the treatment based on
the level of the cleaved HMWK determined in step (iv), wherein if
the level of the cleaved HMWK is equal to or less than a reference
value, the treatment is effective.
19. The immunoassay of claim 10, further comprising identifying a
treatment for the subject based on the level of the cleaved
HMWK.
20. The immunoassay of claim 10, further comprising identifying the
subject as a candidate for a treatment of a disease based on the
level of the cleaved HMWK.
21. The immunoassay of claim 10, wherein the human subject has one
or more symptom of a disease mediated by pKal, wherein the symptom
is selected from the group consisting of Erythema marginatum;
airway blockage; abdominal cramping; vomiting; dehydration;
diarrhea; pain; shock; and swelling in the arms, legs, lips, eyes,
tongue, intestines and/or throat.
22. The immunoassay of claim 21, wherein the disease is hereditary
angioedema (HAE).
23. The immunoassay of claim 10, wherein the human subject has one
or more symptoms of hereditary angioedema (HAE), and wherein the
immunoassay further comprises assessing the risk of disease attack
in the subject, wherein if the level of the cleaved HMWK is greater
than a reference value, there is an indication of the risk of
disease attack.
24. The immunoassay of claim 23, further comprising administering a
therapeutic agent to the subject, if the subject is at risk of
disease attack.
25. The immunoassay of claim 1, wherein the antibody comprises a
heavy chain variable domain comprising
EVQLLESGGGLVQPGGSLRLSCAASGFTFSFYVMVWVRQAPGKGLEWVSGISPSGGNT
AYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARKLFYYDDTKGYFDFWGQ GTLVTVSS
(SEQ ID NO: 4) and a light chain variable domain that comprises
comprising
QYELTQPPSASGTPGQRVTLSCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPSGVP
DRFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLNGRVFGGGTKLTVL (SEQ ID NO:
5).
26. An immunoassay for determining a level of a cleaved high
molecular weight kininogen (HMWK), the immunoassay comprising: (i)
providing a support member, on which a first agent that
specifically binds a cleaved HMWK is immobilized; (ii) contacting
the support member of (i) with a biological sample suspected of
containing the cleaved HMWK; (iii) contacting the support member
obtained in (ii) with a second agent that binds HMWK, wherein the
second agent is conjugated to a label; and (iv) detecting a signal
released from the label of the second agent that is bound to the
support member to determine the level of the cleaved HMWK in the
biological sample; and wherein step (ii) is performed in the
presence of ZnCl.sub.2.
27. The immunoassay of claim 26, wherein the first agent is an
antibody comprising a heavy chain complementarity determining
region (CDR) 1 sequence FSFYVMV, a heavy chain CDR2 sequence
GISPSGGNTAYADSVK, and a heavy chain CDR3 sequence KLFYYDDTKGYFDF
and a light chain CDR1 sequence SGSSSNIGSNYVY, a light chain CDR2
sequence RNNQRPS, and a light chain CDR3 sequence AWDDSLNGRV.
28. The immunoassay of claim 27, wherein the antibody comprises a
heavy chain variable domain comprising
EVQLLESGGGLVQPGGSLRLSCAASGFTFSFYVMVWVRQAPGKGLEWVSGISPSGGNT
AYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARKLFYYDDTKGYFDFWGQ GTLVTVSS
(SEQ ID NO: 4) and a light chain variable domain that comprises
comprising
QYELTQPPSASGTPGQRVTLSCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPSGVP
DRFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLNGRVFGGGTKLTVL (SEQ ID NO:
5).
29. The immunoassay of claim 26, wherein the support member is a
96-well plate.
30. The immunoassay of claim 26, wherein, prior to step (ii), the
support member of (i) is incubated with a blocking buffer.
31. The immunoassay of claim 26, wherein the second agent is a
polyclonal antibody, a monoclonal antibody, or a mixture of two or
more monoclonal antibodies that bind to HMWK.
32. The immunoassay of claim 26, wherein the label is a signal
releasing agent, wherein the signal releasing agent is a dye or
fluorophore.
33. The immunoassay of claim 26, wherein the label is a member of a
receptor-ligand pair and the immunoassay further comprises, prior
to step (iv), contacting the second agent in (iii) that is bound to
the support member, with the other member of the receptor-ligand
pair, wherein the other member is conjugated to a signal releasing
agent.
34. The immunoassay of claim 33, wherein the receptor-ligand pair
is biotin and streptavidin.
35. The immunoassay of claim 26, wherein the immunoassay is a
Western blot assay, an enzyme-linked immunosorbent assay (ELISA),
or a lateral flow assay.
36. The immunoassay of claim 26, wherein the biological sample is
obtained from a human subject.
37. The immunoassay of claim 36, wherein the biological sample is a
serum sample or plasma sample, which is processed from a blood
sample collected in an evacuated blood collection tube comprising
one or more protease inhibitors.
38. The immunoassay of claim 36, wherein the human subject has a
disease and wherein the immunoassay further comprises determining
whether the disease is mediated by plasma kallikrein (pKal) based
on the level of the cleaved HMWK determined in step (iv), wherein
if the level of the cleaved HMWK is greater than a reference value,
the disease is mediated by pKal.
39. The immunoassay of claim 36, further comprising determining
whether the human subject has or is at risk for a disease mediated
by pKal, wherein if the level of the cleaved HMWK is greater than a
reference value, the subject is identified as having or at risk of
having the disease.
40. The immunoassay of claim 39, further comprising administering
to the subject an effective amount of a therapeutic agent for
treating the disease, if the subject is identified as having the
disease.
41. The immunoassay of claim 40, wherein the therapeutic agent is a
plasma kallikrein (pKal) inhibitor, a bradykinin 2 receptor (B2R)
inhibitor, and/or a C1 esterase inhibitor.
42. The immunoassay of claim 41, wherein the pKal inhibitor is an
anti-pKal antibody or an inhibitory peptide.
43. The immunoassay of claim 41, wherein the therapeutic agent is
lanadelumab, ecallantide, icatibant, or human plasma-derived
C1-INH.
44. The immunoassay of claim 36, wherein the human subject is on a
treatment of a disease mediated by pKal, and wherein the method
further comprises assessing the efficacy of the treatment based on
the level of the cleaved HMWK determined in step (iv), wherein if
the level of the cleaved HMWK is equal to or less than a reference
value, from that of a control sample being indicative of the
treatment is effective.
45. The immunoassay of claim 36, further comprising identifying a
treatment for the subject based on the level of the cleaved
HMWK.
46. The immunoassay of claim 36, further comprising identifying the
subject as a candidate for a treatment of a disease based on the
level of the cleaved HMWK.
47. The immunoassay of claim 36, wherein the human subject has one
or more symptom of a disease mediated by pKal, wherein the symptom
is selected from the group consisting of Erythema marginatum;
airway blockage; abdominal cramping; vomiting; dehydration;
diarrhea; pain; shock; and swelling in the arms, legs, lips, eyes,
tongue, intestines and/or throat.
48. The immunoassay of claim 47, wherein the disease is hereditary
angioedema (HAE).
49. The immunoassay of claim 36, wherein the human subject has one
or more symptoms of hereditary angioedema (HAE), and wherein the
immunoassay further comprises assessing the risk of disease attack
in the subject, wherein if the level of the cleaved HMWK is greater
than a reference value, there is an indication of the risk of
disease attack.
50. The immunoassay of claim 49, further comprising administering a
therapeutic agent to the subject, if the subject is at risk of
disease attack.
Description
BACKGROUND OF PRESENT DISCLOSURE
Kininogens are precursors of kinin, such as bradykinin and
kallidin. There are two types of human kininogens, high
molecular-weight kininogen (HMWK) and low molecular-weight
kininogen (LMWK), which are splicing variants. HMWK acts mainly as
a cofactor on coagulation and inflammation and is the preferred
substrate for plasma kallikrein (pKal)-mediated bradykinin
generation.
Plasma kallikrein (pKal) is the primary bradykinin-generating
enzyme in the circulation. The activation of pKal occurs via the
contact system which has been linked to disease pathology
associated with hereditary angioedema (HAE). pKal cleaves HMWK (a
single-chain polypeptide) to produce bradykinin and a cleaved form
HMWK, which contains two polypeptide chains held together by a
disulfide bond. Cugno et al., Blood (1997) 89:3213-3218.
Cleaved HMWK increased to about 47% of total kininogen during a
hereditary angioedema (HAE) attack. Cugno et al., Blood (1997)
89:3213-3218, making it a biomarker for monitoring HAE attack. It
is therefore of interest to develop sensitive and reliable assays
for detecting the level of cleaved HMWK in biological samples.
SUMMARY OF PRESENT DISCLOSURE
Some aspects of the present disclosure provide an immunoassay for
detecting a cleaved high molecular weight kininogen (HMWK) with
high sensitivity and specificity. The method comprises (i)
providing a support member on which a first agent (e.g., an
antibody such as 559B-M004-B04) that specifically binds a cleaved
HMWK is attached; (ii) contacting the support member of (i) with a
biological sample suspected of containing a cleaved HMWK; (iii)
contacting the support member obtained in (ii) with a second agent
that binds HMWK, wherein the second agent is conjugated to a label;
and (iv) detecting a signal released from the label of the second
agent that is bound to the support member, directly or indirectly,
to determine the level of the cleaved HMWK in the biological
sample. In some instances, step (ii) may be performed in the
presence of ZnCl.sub.2.
In some embodiments, prior to step (ii), the support member of (i)
is incubated with a blocking buffer.
In some embodiments, the second agent is a polyclonal antibody, a
monoclonal antibodies, or a mixture of two or more monoclonal
antibodies that bind to HMWK. The two or more monoclonal antibodies
in the mixture may bind to different epitopes in HMWK. In some
embodiments, the label is a signal releasing agent. In some
embodiments, the label is a member of a receptor-ligand pair. In
that case, the immunoassay may further comprise, prior to step
(iv), contacting the second agent in (iii), which is immobilized on
the support member, with the other member of the receptor-ligand
pair, wherein the other member is conjugated to a signal releasing
agent. In one example, the receptor-ligand pair is biotin and
streptavidin.
Another aspect of the present disclosure provides methods for
detecting a cleaved high molecular kininogen (HMWK) in a sample,
the method comprising (i) contacting a sample suspected of
containing a cleaved HMWK with any of the antibodies described
herein (e.g. 559B-M004-B04); (ii) measuring a complex of the
cleaved HMWK and the antibody formed in step (i); and (iii)
determining the level of the cleaved HMWK in the sample based on
the result of step (ii). In some embodiments, step (i) is performed
in the presence of ZnCl.sub.2. In some embodiments, step (i) is
performed using an enzyme-linked immunosorbent assay (ELISA) or an
immunoblotting assay.
In any of the methods described herein, the sample may be a
biological sample obtained from a subject (e.g., a human patient),
such as a serum sample of a plasma sample. In some embodiments, the
method further comprises collecting the sample into an evacuated
blood collection tube, which comprises one or more protease
inhibitors.
Any of the assay methods (e.g., immunoassays) described herein may
be a ELISA assay, a Western blot assay, or lateral flow assay.
In some embodiments, the biological sample is obtained from a
subject (e.g., a human patient) having a disease. The assay method
may further comprise determining whether the disease is mediated by
plasma kallikrein based on the level of the cleaved HMWK, a
deviation of the level of the cleaved HMWK in the sample from that
of a control sample being indicative that the disease is mediated
by plasma kallikrein.
Any of the assay methods described herein may further comprise
identifying patients with diseases or disorders mediated by plasma
kallikrein, or evaluating the efficacy of a treatment of the
disease or disorder based on the levels of cleaved HMWK. In some
embodiments, the method may further comprises administering to the
subject an effective amount of a therapeutic agent, such as a
plasma kallikrein (pKal) inhibitor, a bradykinin 2 receptor (B2R)
inhibitor, and/or a C1 esterase inhibitor, for treating the
disorder, if the subject is identified as having the disorder. In
some embodiments the pKal inhibitor is an anti-pKal antibody. In
some embodiments, the therapeutic agent is lanadelumab,
ecallantide, icatibant, or human plasma-derived C1 esterase
inhibitor.
In some embodiments, the subject is a human patient who is on a
treatment for the disorder, and wherein the method further
comprises assessing the efficacy of the treatment based on the
level of the cleaved HMWK determining in step (iii), a deviation of
the level of the cleaved HMWK in the sample from the subject from
that of a control sample being indicative of the treatment
efficacy. In some embodiments, the method further comprises
identifying a suitable treatment for the subject based on the level
of the cleaved HMWK. In some embodiments, the method further
comprises identifying the subject as a candidate for a treatment of
the disease based on the level of the cleaved HMWK.
In some embodiments, the human patient has a history of the disease
(e.g., HAE). In some embodiments, the method further comprises
assessing the risk of disease attack in the subject based on the
level of the cleaved HMWK, a deviation of the level of the cleaved
HMWK in the sample from the subject from that of a control sample
being indicative of the risk of disease attack. In some
embodiments, the method further comprises administering a
therapeutic agent to the subject, if the subject is at risk of
disease attack.
In another aspect, a kit is provided for detecting a cleaved high
molecular weight kininogen (HMWK), the kit comprising a first agent
(e.g., an antibody as described herein) that specifically binds a
cleaved HMWK. In some embodiments, the kit further comprises a
second agent that binds HMWK, a support member, or both, and
optionally instructions for detecting the cleaved HMWK. In some
examples, the support member is a 96-well plate.
In another aspect of the disclosure, an isolated antibody is
provided, which specifically binds a cleaved high molecular weight
kininogen (HMWK). In some embodiments, the antibody binds the same
epitope as 559B-M004-B04 or competes against 559B-M004-B04 for
binding to the cleaved HMWK. In some embodiments, the antibody
comprises the same heavy chain and light chain complementary
determining regions as 559B-M004-B04, e.g., the same heavy chain
and light variable regions as 559B-M004-B04. In one example, the
antibody is 559B-M004-B04.
Any of the antibodies specific to a cleaved HMWK as described
herein can be used in a method for detecting a cleaved high
molecular kininogen (HMWK) in a sample. Such a method may comprise
(i) contacting a sample suspected of containing a cleaved HMWK with
the antibody; (ii) measuring a complex of the cleaved HMWK and the
antibody formed in step (i); and determining the level of the
cleaved HMWK in the sample based on the result of step (ii). In
some embodiments, the sample is a biological sample such as a serum
sample or a plasma sample obtained from a human subject. The result
obtained from this method may be relied on to determine the risk of
a subject from whom the sample is obtained for developing a
disorder mediated by plasma kallikrein such as HAE. In some
instances, step (i) can be performed in the presence of
ZnCl.sub.2.
Any of the immunoassay methods described herein can be in Western
blot format or ELISA format.
In yet another aspect, an isolated antibody is provided that binds
both intact high molecular weight kininogen (HMWK) and a cleaved
HMWK.
In some embodiments, the antibody that binds both intact and
cleaved HMWK does not bind to low molecular weight kininogen
(LMWK). In some embodiments, the antibody binds the same epitope as
559B-M0067-E02, 559B-M0039-G07, 559B-M0044-E09, 559B-M0003-008,
559B-M0039-H06, 559B-M0039-D08, 559B-M0068-C07, 559B-M0021-G11,
559B-M0061-G06, 559B-M0036-G12, 559B-M0042-E06, 559B-M0070-H10,
559B-M0068-D01, or 559B-M0004-E08. In some embodiments, the
antibody competes against 559B-M0067-E02, 559B-M0039-G07,
559B-M0044-E09, 559B-M0003-C08, 559B-M0039-H06, 559B-M0039-D08,
559B-M0068-C07, 559B-M0021-G11, 559B-M0061-G06, 559B-M0036-G12,
559B-M0042-E06, 559B-M0070-H10, 559B-M0068-D01, or 559B-M0004-E08
for binding to the intact HMWK and/or the cleaved HMWK.
In some embodiments, the antibody comprising the same heavy chain
and light chain CDRs as 559B-M0067-E02, 559B-M0039-G07,
559B-M0044-E09, 559B-M0003-C08, 559B-M0039-H06, 559B-M0039-D08,
559B-M0068-C07, 559B-M0021-G11, 559B-M0061-G06, 559B-M0036-G12,
559B-M0042-E06, 559B-M0070-H10, 559B-M0068-D01, or 559B-M0004-E08.
In some examples, the antibody is selected from the group
consisting of 559B-M0067-E02, 559B-M0039-G07, 559B-M0044-E09,
559B-M0003-C08, 559B-M0039-H06, 559B-M0039-D08, 559B-M0068-C07,
559B-M0021-G11, 559B-M0061-G06, 559B-M0036-G12, 559B-M0042-E06,
559B-M0070-H10, 559B-M0068-D01, and 559B-M0004-E08.
In other embodiments, the antibody that binds both intact and
cleaved HMWK also binds LMWK. In some embodiments, the antibody
binds the same epitope as 559B-M0069-C09, 559B-M0038-F04,
559B-M0044-C05, 559B-M0047-H01, 559B-M0019-E12, 559B-X0004-B05,
559B-M0048-D12, 559B-M0053-G01, 559B-M0038-H03, 559B-M0017-H08,
559B-M0035-F05, 559B-M0035-H09, 559B-M0043-C06, 559B-M0003-A08,
559B-M0054-B11, 559B-M0067-G11, 559B-M0064-H02, or 559B-M0065-B10.
In some embodiments, the antibody competes against 559B-M0069-C09,
559B-M0038-F04, 559B-M0044-C05, 559B-M0047-H01, 559B-M0019-E12,
559B-X0004-B05, 559B-M0048-D12, 559B-M0053-G01, 559B-M0038-H03,
559B-M0017-H08, 559B-M0035-F05, 559B-M0035-H09, 559B-M0043-C06,
559B-M0003-A08, 559B-M0054-B11, 559B-M0067-G11, 559B-M0064-H02, or
559B-M0065-B10 for binding to the intact HMWK, the cleaved HMWK,
and/or the LMWK.
In some embodiments, the antibody comprises the same heavy chain
and light chain CDRs as 559B-M0069-C09, 559B-M0038-F04,
559B-M0044-C05, 559B-M0047-H01, 559B-M0019-E12, 559B-X0004-B05,
559B-M0048-D12, 559B-M0053-G01, 559B-M0038-H03, 559B-M0017-H08,
559B-M0035-F05, 559B-M0035-H09, 559B-M0043-C06, 559B-M0003-A08,
559B-M0054-B11, 559B-M0067-G11, 559B-M0064-H02, or 559B-M0065-B10.
In some examples, the antibody is selected from the group
consisting of 559B-M0069-C09, 559B-M0038-F04, 559B-M0044-C05,
559B-M0047-H01, 559B-M0019-E12, 559B-X0004-B05, 559B-M0048-D12,
559B-M0053-G01, 559B-M0038-H03, 559B-M0017-H08, 559B-M0035-F05,
559B-M0035-H09, 559B-M0043-C06, 559B-M0003-A08, 559B-M0054-B11,
559B-M0067-G11, 559B-M0064-H02, and 559B-M0065-B10.
The details of one or more embodiments of the disclosure are set
forth in the description below. Other features or advantages of the
present disclosure will be apparent from the following drawings and
detailed description of several embodiments, and also from the
appended claims.
BRIEF DESCRIPTION OF DRAWINGS
The following drawings form part of the present specification and
are included to further demonstrate certain aspects of the present
disclosure, which can be better understood by reference to one or
more of these drawings in combination with the detailed description
of specific embodiments presented herein.
FIG. 1 is a graph showing binding of 559B-M0004-B04 to intact HMWK
(dark gray bars) or cleaved HMWK (light gray bars) under the
indicated ELISA conditions.
FIG. 2 presents graphs showing binding of various Fab clones to
intact 1-chain (intact) HMWK, 2-chain (cleaved) HMWK, or LMWK. A:
Fab clones identified using the phage display screening methods
described herein. Intact HWMK is shown in dark gray bars, cleaved
HMWK in light gray bars, and LMWK in medium gray bars. B: binding
for several example Fab clones. LWMK is shown in dark gray bars,
intact HMWK in light gray bars, and cleaved HWMK in medium gray
bars.
FIG. 3 is a graph showing specificity of 559B-M0004-B04 towards
intact HMWK, cleaved HMWK, or LMWK. Purified cleaved HMWK was
spiked into SBT assay buffer (circles) or HMWK-deficient plasma
(squares). Purified intact HMWK was spiked into SBT assay buffer
(triangles). Purified LMWK was spiked into SBT assay buffer
(diamonds). The y-axis presents the ELISA signal in absorbance
units, and the x-axis presents the concentration of kininogen in
.mu.g/mL.
FIG. 4 is a graph showing detection of 2-Chain HMWK (cleaved HMWK)
in plasma or assay buffer. Purified cleaved HMWK was spiked into
SBT assay buffer (open circles), SBT assay buffer and analyzed in
the presence of 10% plasma (squares), or HMWK-deficient plasma and
analyzed in the presence of 10% plasma (triangles). Purified
cleaved HMWK was also spiked into assay buffer and analyzed in the
presence of 2.5% plasma (diamonds) or HMWK deficient plasma and
analyzed in the presence of 2.5% plasma (closed circles). The
y-axis presents the ELISA signal in absorbance units, and the
x-axis presents the concentration of kininogen in .mu.g/mL.
FIG. 5 is a graph showing levels of cleaved HMWK in the indicated
human plasma samples prior to and after contact system activation.
A: prior to and after contact system activation with FXIIa or
ellagic acid. B: prior to and after contact system activation with
FXIIa, pKal, or ellagic acid.
FIG. 6 is a graph showing levels of cleaved HMWK in plasma samples
from 12 normal human donors prior to and after activation of the
contact system with ellagic acid.
FIG. 7 presents graphs showing levels of cleaved HMWK following
inhibition with a pKal inhibitor. A: inhibition with
landadelumab/DX-2930 or C1-INH prior to contact system activation
with ellagic acid. B: inhibition of pooled sodium citrate plasma
samples with landadelumab/DX-2930 prior to contact system
activation with 10 nM FXIIa.
FIG. 8 is a graph showing cleaved HMWK generation at the indicated
time points following contact system activation with FXIIa or
ellagic acid.
FIG. 9 is a graph showing levels of 2-chain HMWK in plasma samples
from normal subjects and subjects having HAE.
FIG. 10 is photo showing results obtained from a HMWK Western blot
analysis, which are consistent with the results obtained from the
2-Chain HMWK ELISA assay described herein. Human citrated plasma
samples (normal plasma, FXII-deficient plasma, and
prekallikrein-deficient plasma) were probed with a mouse monoclonal
anti-HMWK light chain antibody followed by a goat anti-mouse
detection antibody. The analyzed plasma samples were either
untreated or activated with 100 nM pKal, 10 nM FXIIa, or 10%
ellagic acid.
FIG. 11 is a graph showing that the addition of ZnCl.sub.2 to
either citrated or EDTA plasma samples increased the signal of the
2-Chain HMWK in an ELISA assay. The x-axis shows the concentration
of ZnCl.sub.2 in the assay well after a 40-fold dilution.
FIG. 12 presents schematics of the discovery and development of
assays using the antibodies described herein. A: schematic of the
phage display methods used to discover 2-chain HMWK binding
antibodies. B: an example sandwich ELISA assay in which the 2-chain
HMWK specific antibody/Fab (e.g., 559B-M0004-B04) is immobilized in
96-well plates to capture 2-chain HMWK in citrated plasma, followed
by washing and detection with an anti-HMWK antibody conjugated to a
label (anti-HMWK-HRP).
FIG. 13 is a graph showing results from a 2-chain HMWK sandwich
ELISA standard curve, in which citrated plasma samples were spiked
with 2-chain HMWK (10% final dilution).
FIG. 14 shows the identification of 2-chain HMWK-specific
antibodies by phage display selection and screening. A: plots the
ratio of the result of a 2-chain HWMK binding assay to a LMWK
binding assay on the y-axis compared to the ratio of the result of
a 2-chain HMWK binding assay to a 1-chain HMWK binding assay on the
x-axis for each antibody (Fab) tested. Recombinant Fab fragments
were passively immobilized onto 384-well plates prior to addition
of biotinylated 2-chain HMWK, 1-chain HMWK, or LMWK, followed by
streptavidin-HRP. B: shows binding to 1-chain HMWK, 2-chain HMWK,
or LMWK for the indicated isolated Fab fragments.
FIG. 15 is a graph showing competition of 2-chain HMWK and
kininogen peptides (HKH20 and GCP28) for binding to
559B-M0004-B04.
FIG. 16 is a graph showing a standard curve for an optimized
sandwich ELISA for the detection of 2-chain HMWK in human plasma
samples.
FIG. 17 presents graphs of Western blotting analyses comparing the
level of 2-chain HMWK in citrated plasma samples from healthy
subjects and HAE patients. A: scatter plot comparing the percent
2-chaim HMWK in samples from healthy subjects ("HV") and HAE
patients between HAE attacks ("Basal") and during an HAE attack
("Attack"). B: ROC (receiver operating characteristic) analysis
comparing the sensitivity and specificity for the detection of HAE
basal samples versus samples from healthy subjects (AUC=0.977). C:
ROC analysis comparing the sensitivity and specificity for the
detection of HAE attack samples versus samples from healthy
subjects (AUC=1). D: ROC analysis comparing the sensitivity and
specificity for the detection of HAE attack samples versus HAE
basal samples (AUC=0.625).
FIG. 18 presents graphs of Western blotting analyses comparing the
level of 2-chain HMWK in SCAT169 plasma samples from healthy
subjects and HAE patients. A: scatter plot comparing the percent
2-chaim HMWK in samples from healthy subjects ("HV") and HAE
patients between HAE attacks ("Basal") and during an HAE attack
("Attack"). B: ROC analysis comparing the sensitivity and
specificity for the detection of HAE basal samples versus samples
from healthy subjects (AUC=0.915). C: ROC analysis comparing the
sensitivity and specificity for the detection of HAE attack samples
versus samples from healthy subjects (AUC=0.967). D: ROC analysis
comparing the sensitivity and specificity for the detection of HAE
attack samples versus HAE basal samples (AUC=0.597).
FIG. 19 presents graphs of ELISA analyses comparing the level of
2-chain HMWK in citrated plasma samples from healthy subjects and
HAE patients. A: scatter plot comparing the percent 2-chaim HMWK in
samples from healthy subjects ("HV") and HAE patients between HAE
attacks ("Basal") and during an HAE attack ("Attack"). B: ROC
analysis comparing the sensitivity and specificity for the
detection of HAE basal samples versus samples from healthy subjects
(AUC=0.795). C: ROC analysis comparing the sensitivity and
specificity for the detection of HAE attack samples versus samples
from healthy subjects (AUC=0.866). D: ROC analysis comparing the
sensitivity and specificity for the detection of HAE attack samples
versus HAE basal samples (AUC=0.709).
FIG. 20 presents graphs of ELISA analyses comparing the level of
2-chain HMWK in SCAT169 samples from healthy subjects and HAE
patients. A: scatter plot comparing the percent 2-chaim HMWK in
samples from healthy subjects ("HV") and HAE patients between HAE
attacks ("Basal") and during an HAE attack ("Attack"). B: ROC
analysis comparing the sensitivity and specificity for the
detection of HAE basal samples versus samples from healthy subjects
(AUC=0.999). C: ROC analysis comparing the sensitivity and
specificity for the detection of HAE attack samples versus samples
from healthy subjects (AUC=1). D: ROC analysis comparing the
sensitivity and specificity for the detection of HAE attack samples
versus HAE basal samples (AUC=0.8176).
DETAILED DESCRIPTION OF PRESENT DISCLOSURE
Plasma kallikrein (PKal) is a serine protease component of the
contact system and is the primary bradykinin-generating enzyme in
the circulation. The contact system is activated by either factor
XIIa (the active form of Factor XII or FXII) upon exposure to
foreign or negatively charged surfaces or on endothelial cell
surfaces by prolylcarboxypeptidases (Sainz I. M. et al., Thromb
Haemost 98, 77-83, 2007). Activation of the plasma kallikrein
amplifies intrinsic coagulation via its feedback activation of
factor XII and proteolytically cleaves the kininogen precursor,
high molecular weight kininogen (HMWK), releasing the
proinflammatory nonapeptide bradykinin and a cleaved HMWK, which
contains two polypeptide chains linked by a disulfide bond (also
known as 2-chain HMWK).
As the primary kininogenase in the circulation, plasma kallikrein
is largely responsible for the generation of bradykinin in the
vasculature. A genetic deficiency in the C1-inhibitor protein
(C1-INH) leads to hereditary angioedema (HAE). Patients with HAE
suffer from acute attacks of painful edema often precipitated by
unknown triggers (Zuraw B. L. et al., N Engl J Med 359, 1027-1036,
2008). Through the use of pharmacological agents or genetic studies
in animal models, the plasma kallikrein-kinin system (plasma KKS)
has been implicated in various diseases.
The level of cleaved HMWK was found to be elevated in HAE attack,
as well as in other pKal-associated disorders. Thus, cleaved HMWK
can serve as a biomarker for monitoring disease development and/or
treatment efficacy. However, the art lacks suitable agents and/or
suitable assays that can effectively distinguish intact HMWK from
its cleaved version.
The present disclosure is based, at least in part, on the
development of specific immunoassays that allows for detection of
cleaved HMWK with high specificity and sensitivity. It was observed
that a Sandwich ELISA in which an agent that specifically binds
cleaved HMWK is immobilized on a support member (e.g., a multi-well
plate) unexpectedly enhanced detection efficiency as compared to
the setting of ELISA in which the antigen (in this case, the
cleaved HMWK) is immobilized on the support member. Further, it was
observed, unexpectedly, that using the LowCross blocking buffer
(containing casein), rather than a blocking buffer containing
bovine serum album (BSA), enhanced detection specificity and
sensitivity during the initial screening to discover antibodies
specific for cleaved HMWK. Moreover, the detection specificity and
sensitivity was further enhanced when a 96-well plate was used, as
compared with a 384-well plate. The present disclosure is also
based on, at least in part, the isolation of antibodies that
specifically bind a cleaved HMWK.
Accordingly, provided herein are immunoassays for detecting the
presence or measuring the level of a cleaved HMWK in a biological
sample suspected of containing HMWK species, using an agent (e.g.,
an antibody) that specifically binds a cleaved HMWK (e.g., the
cleaved HMWK having a molecular weight of 46 kDa). Given the
correlation between the level of cleaved HMWK and disorders
associated with or mediated by pKal (e.g., HAE), the imunoassays
described herein can be applied to identify patients who are at
risk of such diseases, to monitor disease progression, and/or to
monitor efficacy of a treatment against such a disorder.
I. Immunoassays for Specific Detection of Cleaved HMWK
One aspect of the present disclosure relates to immunoassays for
detecting cleaved HMWK with high sensitivity and specificity. Such
immunoassays may involve a Sandwich ELISA in which an agent that
specifically binds a cleaved HMWK is immobilized on a support
member, which can be a 96-well plate. The immunoassays described
herein allows for selective detection of cleaved HMWK in biological
samples, e.g., serum samples or plasma samples, which may contain
both intact and cleaved HMWK, as well as LMWK.
(i) High Molecular-Weight Kininogen
High molecular-weight kininogen (HMWK) exists in the plasma as a
single polypeptide (1-chain) multi-domain (domains 1-6) protein
with a molecular weight of approximately 110 kDa, referred to
herein as intact HWMK. The human gene encoding HMWK is kininogen 1
(KNG1). KNG1 is transcribed and alternatively spliced to form mRNAs
that encode either HMWK or low molecular weight kininogen (LMWK).
An exemplary protein sequence of HMWK is provided below:
TABLE-US-00001 >gi|156231037|ref|NP_001095886.1| kininogen-1
isoform 1 precursor [Homo sapiens] (SEQ ID NO: 1)
MKLITILFLCSRLLLSLTQESQSEEIDCNDKDLFKAVDAALKKYNSQNQS
NNQFVLYRITEATKTVGSDTFYSFKYEIKEGDCPVQSGKTWQDCEYKDAA
KAATGECTATVGKRSSTKFSVATQTCQITPAEGPVVTAQYDCLGCVHPIS
TQSPDLEPILRHGIQYFNNNTQHSSLFMLNEVKRAQRQVVAGLNFRITYS
IVQTNCSKENFLFLTPDCKSLWNGDTGECTDNAYIDIQLRIASFSQNCDI
YPGKDFVQPPTKICVGCPRDIPTNSPELEETLTHTITKLNAENNATFYFK
IDNVKKARVQVVAGKKYFIDEVARETTCSKESNEELTESCETKKLGQSLD
CNAEVYVVPWEKKIYPTVNCQPLGMISLMKRPPGFSPFRSSRIGEIKEET
TVSPPHTSMAPAQDEERDSGKEQGHTRRHDWGHEKQRKHNLGHGHKHERD
QGHGHQRGHGLGHGHEQQHGLGHGHKFKLDDDLEHQGGHVLDHGHKHKHG
HGHGKHKNKGKKNGKHNGWKTEHLASSSEDSTTPSAQTQEKTEGPTPIPS
LAKPGVTVTFSDFQDSDLIATMMPPISPAPIQSDDDWIPDIQIDPNGLSF
NPISDFPDTTSPKCPGRPWKSVSEINPTTQMKESYYFDLTDGLS
Intact HMWK, also referred to herein as "intact kininogen," can be
assayed, for example, using coagulant or immunological methods,
e.g., radioimmunoassay (see, e.g., Kerbiriou-Nabias, D. M., Br J
Haematol, 1984, 56(2):2734-86). A monoclonal antibody to the light
chain of human HMWK is known. See, e.g., Reddigari, S. R. &
Kaplan, A. P., Blood, 1999, 74:695-702. An assay for HMWK that
relies on a chromogenic substrate can also be used. See, e.g.,
Scott, C. F. et al. Thromb Res, 1987, 48(6):685-700; Gallimore, M.
J. et al. Thromb Res, 2004, 114(2):91-96.
HMWK is cleaved by pKal within domain 4 to release the 9 amino
acid, pro-inflammatory peptide bradykinin, and a 2-chain form of
HMWK, referred to herein as cleaved HMWK. The 2 chains of HMWK are
the heavy chain, which contains domains 1-3, and the light chain,
which contains domains 5 and 6, joined by a disulfide bond. Upon
initial cleavage of intact HMWK, the heavy and light chains have a
molecular weight of approximately 65 kDa and 56 kDa, respectively.
Further proteolytic processing results in generation of a 46 kDa
light chain.
Exemplary sequences of the heavy and light chains of cleaved
kininogen are provided below.
TABLE-US-00002 > cleaved kininogen-1 heavy chain (SEQ ID NO: 2)
QESQSEEIDCNDKDLFKAVDAALKKYNSQNQSNNQFVLYRITEATKTVGS
DTFYSFKYEIKEGDCPVQSGKTWQDCEYKDAAKAATGECTATVGKRSSTK
FSVATQTCQITPAEGPVVTAQYDCLGCVHPISTQSPDLEPILRHGIQYFN
NNTQHSSLFMLNEVKRAQRQVVAGLNFRITYSIVQTNCSKENFLFLTPDC
KSLWNGDTGECTDNAYIDIQLRIASFSQNCDIYPGKDFVQPPTKICVGCP
RDIPTNSPELEETLTHTITKLNAENNATFYFKIDNVKKARVQVVAGKKYF
IDFVARETTCSKESNEELTESCETKKLGQSLDCNAEVYVVPWEKKIYPTV NCQPLGMISLMK
> cleaved kininogen-1 light chain (SEQ ID NO: 3)
SSRIGEIKEETTVSPPHTSMAPAQDEERDSGKEQGHTRRHDWGHEKQRKH
NLGHGHKHERDQGHGHQRGHGLGHGHEQQHGLGHGHKFKLDDDLEHQGGH
VLDHGHKHKHGHGHGKHKNKGKKNGKHNGWKTEHLASSSEDSTTPSAQTQ
EKTEGPTPIPSLAKPGVTVTFSDFQDSDLIATMMPPISPAPIQSDDDWIP
DIQIDPNGLSFNPISDFPDTTSPKCPGRPWKSVSEINPTTQMKESYYFDL TDGLS
(ii) Antibodies Specific to Cleaved HMWK
The immunoassays described herein may use any agent that can
specifically bind a cleaved HMWK, for example, an agent that
recognizes a neoepitope on cleaved HMWK that is not present on
intact HMWK. In some embodiments, the cleaved HMWK-binding agent is
an antibody.
An antibody (interchangeably used in plural form) is an
immunoglobulin molecule capable of specific binding to a target,
such as a carbohydrate, polynucleotide, lipid, polypeptide, etc.,
through at least one antigen recognition site, located in the
variable region of the immunoglobulin molecule. As used herein, the
term "antibody" encompasses not only intact (i.e., full-length)
polyclonal or monoclonal antibodies, but also antigen-binding
fragments thereof (such as Fab, Fab', F(ab').sub.2, Fv), single
chain (scFv), mutants thereof, fusion proteins comprising an
antibody portion, humanized antibodies, chimeric antibodies,
diabodies, linear antibodies, single chain antibodies,
multispecific antibodies (e.g., bispecific antibodies) and any
other modified configuration of the immunoglobulin molecule that
comprises an antigen recognition site of the required specificity,
including glycosylation variants of antibodies, amino acid sequence
variants of antibodies, and covalently modified antibodies. An
antibody includes an antibody of any class, such as IgD, IgE, IgG,
IgA, or IgM (or sub-class thereof), and the antibody need not be of
any particular class. Depending on the antibody amino acid sequence
of the constant domain of its heavy chains, immunoglobulins can be
assigned to different classes. There are five major classes of
immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these
may be further divided into subclasses (isotypes), e.g., IgG1,
IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy-chain constant domains
that correspond to the different classes of immunoglobulins are
called alpha, delta, epsilon, gamma, and mu, respectively. The
subunit structures and three-dimensional configurations of
different classes of immunoglobulins are well known.
Any of the antibodies described herein can be either monoclonal or
polyclonal. A "monoclonal antibody" refers to a homogenous antibody
population and a "polyclonal antibody" refers to a heterogeneous
antibody population. These two terms do not limit the source of an
antibody or the manner in which it is made.
An antibody that "specifically binds" a cleaved HMWK or an epitope
thereof is a term well understood in the art, and methods to
determine such specific binding are also well known in the art. A
molecule is said to exhibit "specific binding" if it reacts or
associates more frequently, more rapidly, with greater duration
and/or with greater affinity with a particular target antigen (here
a cleaved HMWK) than it does with alternative targets (e.g., intact
HMWK and/or LMWK). An antibody "specifically binds" to a target
antigen if it binds with greater affinity, avidity, more readily,
and/or with greater duration than it binds to other substances. For
example, an antibody that specifically (or preferentially) binds to
cleaved HMWK or an epitope therein is an antibody that binds this
target antigen with greater affinity, avidity, more readily, and/or
with greater duration than it binds to other antigens (e.g., intact
HMWK or LMWK) or other epitopes in the same antigen. It is also
understood by reading this definition that, for example, an
antibody that specifically binds to a first target antigen may or
may not specifically or preferentially bind to a second target
antigen. As such, "specific binding" or "preferential binding" does
not necessarily require (although it can include) exclusive
binding. Generally, but not necessarily, reference to binding means
preferential binding.
In some embodiments, the antibodies that specifically binds cleaved
HMWK (as well the other antibodies that bind both cleaved and
intact, and optionally LMWK) described herein have a suitable
binding affinity to a cleaved HMWK (or another target antigen as
described herein). As used herein, "binding affinity" refers to the
apparent association constant or K.sub.A. The K.sub.A is the
reciprocal of the dissociation constant (K.sub.D). The antibody
described herein may have a binding affinity (K.sub.D) of at least
10.sup.-5, 10.sup.-6, 10.sup.-7, 10.sup.-8, 10.sup.-9, 10.sup.-10
M, or lower. An increased binding affinity corresponds to a
decreased K.sub.D. Higher affinity binding of an antibody to a
first target relative to a second target can be indicated by a
higher K.sub.A (or a smaller numerical value K.sub.D) for binding
the first target than the K.sub.A (or numerical value K.sub.D) for
binding the second target. In such cases, the antibody has
specificity for the first target (e.g., a protein in a first
conformation or mimic thereof) relative to the second target (e.g.,
the same protein in a second conformation or mimic thereof; or a
second protein). Differences in binding affinity (e.g., for
specificity or other comparisons) can be at least 1.5, 2, 3, 4, 5,
10, 15, 20, 37.5, 50, 70, 80, 91, 100, 500, 1000, 10,000 or
10.sup.5 fold. For example, the binding affinity of an antibody
that specifically binds a cleaved HMWK as described herein may be
10-fold, 100-fold, 10,000-fold, or 10.sub.5-fold higher than the
binding affinity of that antibody to intact HMWK and/or LMWK.
Binding affinity can be determined by a variety of methods
including equilibrium dialysis, equilibrium binding, gel
filtration, ELISA, surface plasmon resonance, or spectroscopy
(e.g., using a fluorescence assay). Exemplary conditions for
evaluating binding affinity are in HBS-P buffer (10 mM HEPES pH7.4,
150 mM NaCl, 0.005% (v/v) Surfactant P20). These techniques can be
used to measure the concentration of bound binding protein as a
function of target protein concentration. The concentration of
bound binding protein ([Bound]) is related to the concentration of
free target protein ([Free]) and the concentration of binding sites
for the binding protein on the target where (N) is the number of
binding sites per target molecule by the following equation:
[Bound]=[N][Free]/(Kd+[Free])
It is not always necessary to make an exact determination of
K.sub.A, though, since sometimes it is sufficient to obtain a
quantitative measurement of affinity, e.g., determined using a
method such as ELISA or FACS analysis, is proportional to K.sub.A,
and thus can be used for comparisons, such as determining whether a
higher affinity is, e.g., 2-fold higher, to obtain a qualitative
measurement of affinity, or to obtain an inference of affinity,
e.g., by activity in a functional assay, e.g., an in vitro or in
vivo assay.
In some embodiments, the antibody that specifically binds to
cleaved HMWK (also referred to as an anti-cleaved HMWK antibody)
binds to the same epitope of a cleaved HMWK as 559B-M004-B04. An
"epitope" refers to the site on a target antigen that is bound by a
binding protein (e.g., an antibody such as a Fab or full length
antibody). The site can be entirely composed of amino acid
components, entirely composed of chemical modifications of amino
acids of the protein (e.g., glycosyl moieties), or composed of
combinations thereof. Overlapping epitopes include at least one
common amino acid residue, glycosyl group, phosphate group, sulfate
group, or other molecular feature. In some cases, the epitope is
linear; in other instances, the epitope is conformational.
A first antibody "binds to the same epitope" as a second antibody
if the first antibody binds to the same site on a target antigen
that the second antibody binds, or binds to a site that overlaps
(e.g., 50%, 60%, 70%, 80%, 90%, or 100% overlap, e.g., in terms of
amino acid sequence or other molecular feature (e.g., glycosyl
group, phosphate group, or sulfate group) with the site that the
second antigen binds.
In some embodiments, the antibody that specifically binds to
cleaved HMWK competes against 559B-M004-B04 for binding to HMWK. A
first antibody "competes for binding" with a second antibody if the
binding of the first antibody to its epitope decreases (e.g., by
10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more) the
amount of the second antibody that binds to its epitope. The
competition can be direct (e.g., the first antibody binds to an
epitope that is the same as, or overlaps with, the epitope bound by
the second antibody), or indirect (e.g., the binding of the first
antibody to its epitope causes a steric change in the target
antigen that decreases the ability of the second antibody to bind
to its epitope).
In some examples, the antibody that specifically binds to cleaved
HMWK comprises a V.sub.H chain that includes a V.sub.H CDR1, a
V.sub.H CDR2, and/or a V.sub.H CDR3 at least 75% (e.g., 80%, 85%,
90%, 95%, 96%, 97%, 98%, or 99%) identical to the corresponding
V.sub.H CDRs of 559B-M004-B04. Alternatively or in addition, the
antibody that specifically binds to cleaved HMWK comprises a
V.sub.L CDR1, a V.sub.L CDR2, and/or a V.sub.L CDR3 at least 75%
(e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) identical to the
corresponding V.sub.L CDRs of 559B-M004-B04. In some embodiments,
the antibody that specifically binds to cleaved HMWK has the same
heavy chain and/or light chain complementarity determining regions
(CDRs) as 559B-M004-B04.
"Complementarity determining regions" or "CDRs" are known in the
art as referring to non-contiguous sequences of amino acids within
antibody variable regions, which confer antigen specificity and
binding affinity. In general, there are three (3) CDRs in each
heavy chain variable region and three (3) CDRs in each light chain
variable region. The precise amino acid sequence boundaries of a
given CDR can be readily determined using any of a number of
well-known schemes, including those described by Kabat et al.
(1991), 5th Ed. Public Health Service, National Institutes of
Health, Bethesda, Md. (the Kabat'' numbering scheme), Al-Lazikani
et al., (1997) JMB 273, 927-948 (the Chothia'' numbering scheme),
MacCallum et al., J. Mol. Biol. 262:732-745 (1996) (the Contact
numbering scheme), Lefranc M P et al., Dev Comp Immunol, 2003
January; 27(1):55-77 (the IMGT numbering scheme), and Honegger A
and Pluckthun A, J Mol Biol, 2001 Jun. 8; 309(3):657-70, (the AHo
numbering scheme).
The boundaries of a given CDR may vary depending on the scheme used
for identification. For example, the Kabat scheme is based
structural alignments, while the Chothia scheme is based on
structural information. The Contact scheme is based on analysis of
complex crystal structures and is similar in many respects to the
Chothia numbering scheme. Thus, unless otherwise specified, the
term "complementary determining region" or "CDR" of a given
antibody should be understood to encompass the complementary
determining region as defined by any of the known schemes described
herein above.
If, determined by the same numbering scheme, an antibody has the
same V.sub.H and/or V.sub.L CDRs as 559B-M004-B04 (as well as other
exemplary antibodies disclosed herein), such an antibody is deemed
as having the same CDRs as 559B-M004-B04 (or the other exemplary
antibodies disclosed herein) and is within the scope of the present
disclosure. For example, such an antibody may have the same V.sub.H
and/or V.sub.L CDRs as clone 559B-M004-B04 as determined by the
Chothia numbering scheme. In another example, an anti-cleaved HMWK
antibody within the scope of the present disclosure may have the
same V.sub.H and/or V.sub.L CDRs as clone 559B-M004-B04, as
determined by the Kabat numbering scheme.
Alternatively or in addition, the anti-cleaved HMWK antibody
comprises a V.sub.H chain at least 75% (e.g., 80%, 85%, 90%, 95%,
96%, 97%, 98%, or 99%) identical to the V.sub.H chain of
559B-M004-B04 and/or a V.sub.L chain at least 75% (e.g., 80%, 85%,
90%, 95%, 96%, 97%, 98%, or 99%) identical to the V.sub.L chain of
559B-M004-B04. In some embodiments, the antibody is
559B-M004-B04.
The "percent identity" of two amino acid sequences is determined
using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci.
USA 87:2264-68, 1990, modified as in Karlin and Altschul Proc.
Natl. Acad. Sci. USA 90:5873-77, 1993. Such an algorithm is
incorporated into the NBLAST and XBLAST programs (version 2.0) of
Altschul, et al. J. Mol. Biol. 215:403-10, 1990. BLAST protein
searches can be performed with the XBLAST program, score=50,
wordlength=3 to obtain amino acid sequences homologous to the
protein molecules of interest. Where gaps exist between two
sequences, Gapped BLAST can be utilized as described in Altschul et
al., Nucleic Acids Res. 25(17):3389-3402, 1997. When utilizing
BLAST and Gapped BLAST programs, the default parameters of the
respective programs (e.g., XBLAST and NB LAST) can be used.
The sequences of the heavy chain variable region and the light
chain variable region of 559B-M004-B04 are shown below. Heavy chain
CDR1, CDR2, and CDR3 sequences and light chain CDR1, CDR2, and CDR3
sequences are underlined and in boldface (identified by one scheme
as an example).
TABLE-US-00003 >559B-R0048-A01 (559B-M0004-B04) Heavy Chain
Amino Acid Sequence (SEQ ID NO: 4)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSFYVMVWVRQAPGKGLEWVSG
ISPSGGNTAYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARKL
FYYDDTKGYFDFWGQGTLVTVSS >559B-R0048-A01 (559B-M0004-B04) Light
Chain Amino Acid Sequence (SEQ ID NO: 5)
QYELTQPPSASGTPGQRVTLSCSGSSSNIGSNYVYWYQQLPGTAPKLLIY
RNNQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLNGRV FGGGTKLTVL
In some instances, the antibody that specifically binds a cleaved
HMWK may contain one or more (e.g., up to 5, up to 3, or up to 1)
conservative mutations in one or more of the heavy chain CDRs, or
one or more of the light chain CDRs in 559B-M0004-B04, e.g., at
positions where the residues are not likely to be involved in
interacting with the cleaved HMWK. As used herein, a "conservative
amino acid substitution" refers to an amino acid substitution that
does not alter the relative charge or size characteristics of the
protein in which the amino acid substitution is made. Variants can
be prepared according to methods for altering polypeptide sequence
known to one of ordinary skill in the art such as are found in
references which compile such methods, e.g. Molecular Cloning: A
Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, or
Current Protocols in Molecular Biology, F. M. Ausubel, et al.,
eds., John Wiley & Sons, Inc., New York. Conservative
substitutions of amino acids include substitutions made amongst
amino acids within the following groups: (a) M, I, L, V; (b) F, Y,
W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.
Antibodies capable of binding to cleaved HMWK (as well as
antibodies capable of binding to intact HMWK and/or LMWK) as
described herein can be made by any method known in the art. See,
for example, Harlow and Lane, (1988) Antibodies: A Laboratory
Manual, Cold Spring Harbor Laboratory, New York.
In some embodiments, antibodies specific to a target antigen (a
cleaved HMWK, the intact HMWK, and/or LMWK) can be made by the
conventional hybridoma technology. The full-length target antigen
or a fragment thereof, optionally coupled to a carrier protein such
as KLH, can be used to immunize a host animal for generating
antibodies binding to that antigen. The route and schedule of
immunization of the host animal are generally in keeping with
established and conventional techniques for antibody stimulation
and production, as further described herein. General techniques for
production of mouse, humanized, and human antibodies are known in
the art and are described herein. It is contemplated that any
mammalian subject including humans or antibody producing cells
therefrom can be manipulated to serve as the basis for production
of mammalian, including human hybridoma cell lines. Typically, the
host animal is inoculated intraperitoneally, intramuscularly,
orally, subcutaneously, intraplantar, and/or intradermally with an
amount of immunogen, including as described herein.
Hybridomas can be prepared from the lymphocytes and immortalized
myeloma cells using the general somatic cell hybridization
technique of Kohler, B. and Milstein, C. (1975) Nature 256:495-497
or as modified by Buck, D. W., et al., In Vitro, 18:377-381 (1982).
Available myeloma lines, including but not limited to X63-Ag8.653
and those from the Salk Institute, Cell Distribution Center, San
Diego, Calif., USA, may be used in the hybridization. Generally,
the technique involves fusing myeloma cells and lymphoid cells
using a fusogen such as polyethylene glycol, or by electrical means
well known to those skilled in the art. After the fusion, the cells
are separated from the fusion medium and grown in a selective
growth medium, such as hypoxanthine-aminopterin-thymidine (HAT)
medium, to eliminate unhybridized parent cells. Any of the media
described herein, supplemented with or without serum, can be used
for culturing hybridomas that secrete monoclonal antibodies. As
another alternative to the cell fusion technique, EBV immortalized
B cells may be used to produce the anti-PKal monoclonal antibodies
described herein. The hybridomas are expanded and subcloned, if
desired, and supernatants are assayed for anti-immunogen activity
by conventional immunoassay procedures (e.g., radioimmunoassay,
enzyme immunoassay, or fluorescence immunoassay).
Hybridomas that may be used as source of antibodies encompass all
derivatives, progeny cells of the parent hybridomas that produce
monoclonal antibodies capable of interfering with the PKal
activity. Hybridomas that produce such antibodies may be grown in
vitro or in vivo using known procedures. The monoclonal antibodies
may be isolated from the culture media or body fluids, by
conventional immunoglobulin purification procedures such as
ammonium sulfate precipitation, gel electrophoresis, dialysis,
chromatography, and ultrafiltration, if desired. Undesired activity
if present, can be removed, for example, by running the preparation
over adsorbents made of the immunogen attached to a solid phase and
eluting or releasing the desired antibodies off the immunogen.
Immunization of a host animal with a target antigen or a fragment
containing the target amino acid sequence conjugated to a protein
that is immunogenic in the species to be immunized, e.g., keyhole
limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean
trypsin inhibitor using a bifunctional or derivatizing agent, for
example maleimidobenzoyl sulfosuccinimide ester (conjugation
through cysteine residues), N-hydroxysuccinimide (through lysine
residues), glutaraldehyde, succinic anhydride, SOCl, or
R1N.dbd.C.dbd.NR, where R and R1 are different alkyl groups, can
yield a population of antibodies (e.g., monoclonal antibodies).
If desired, an antibody (monoclonal or polyclonal) of interest
(e.g., produced by a hybridoma) may be sequenced and the
polynucleotide sequence may then be cloned into a vector for
expression or propagation. The sequence encoding the antibody of
interest may be maintained in vector in a host cell and the host
cell can then be expanded and frozen for future use. In an
alternative, the polynucleotide sequence may be used for genetic
manipulation to improve the affinity (affinity maturation), or
other characteristics of the antibody. It may be desirable to
genetically manipulate the antibody sequence to obtain greater
affinity and/or specificity to the target antigen. It will be
apparent to one of skill in the art that one or more polynucleotide
changes can be made to the antibody and still maintain its binding
specificity to the target antigen.
In other embodiments, fully human antibodies can be obtained by
using commercially available mice that have been engineered to
express specific human immunoglobulin proteins. Transgenic animals
that are designed to produce a more desirable (e.g., fully human
antibodies) or more robust immune response may also be used for
generation of humanized or human antibodies. Examples of such
technology are Xenomouse.RTM. from Amgen, Inc. (Fremont, Calif.)
and HuMAb-Mouse.RTM. and TC Mouse.TM. from Medarex, Inc.
(Princeton, N.J.). In another alternative, antibodies may be made
recombinantly by phage display or yeast technology. See, for
example, U.S. Pat. Nos. 5,565,332; 5,580,717; 5,733,743; and
6,265,150; and Winter et al., (1994) Annu. Rev. Immunol.
12:433-455, and. Alternatively, the phage display technology
(McCafferty et al., (1990) Nature 348:552-553) can be used to
produce human antibodies and antibody fragments in vitro, from
immunoglobulin variable (V) domain gene repertoires from
unimmunized donors.
Antigen-binding fragments of an intact antibody (full-length
antibody) can be prepared via routine methods. For example, F(ab')2
fragments can be produced by pepsin digestion of an antibody
molecule, and Fab fragments that can be generated by reducing the
disulfide bridges of F(ab')2 fragments.
A single-chain antibody can be prepared via recombinant technology
by linking a nucleotide sequence coding for a heavy chain variable
region and a nucleotide sequence coding for a light chain variable
region. Preferably, a flexible linker is incorporated between the
two variable regions. Alternatively, techniques described for the
production of single chain antibodies (U.S. Pat. Nos. 4,946,778 and
4,704,692) can be adapted to produce a phage or yeast scFv library
and scFv clones specific to a PKal can be identified from the
library following routine procedures. Positive clones can be
subjected to further screening to identify those that specifically
bind a target antigen, such as a cleaved HMWK.
In some embodiments, the antibodies specific to a cleaved HMWK (or
to intact HMWK or LMWK) may be isolated from an antibody library,
which may be a synthetic library or a natural library. A natural
antibody library refers to a library derived from a natural source
(e.g., a human donor) following routine practice. A synthetic
antibody library refers to a library the members of which are
designed following predetermined rules (e.g., having a complete
randomized CDR region such as CDRs or a semi randomized CDR region
such as CDR1 or CDR2 of the heavy chain, the light chain, or
both).
In some instances, the antibody library is a display library (e.g.,
a phage display library or a yeast display library). A display
library is a collection of entities; each entity includes an
accessible polypeptide component and a recoverable component that
encodes or identifies the polypeptide component. The polypeptide
component is varied so that different amino acid sequences are
represented. The polypeptide component can be of any length, e.g.,
from three amino acids to over 300 amino acids. A display library
entity can include more than one polypeptide component, for
example, the two polypeptide chains of a sFab. In one exemplary
implementation, a display library can be used to identify proteins
that bind to a cleaved HMWK (as well as other target antigens
described herein). In a selection, the polypeptide component of
each member of the library is probed with a cleaved HMWK (or a
fragment thereof) and if the polypeptide component binds to the
cleaved HMWK, the display library member is identified, typically
by retention on a support. An exemplary illustration for
identifying antibodies specific to cleaved HMWK using a phage
display antibody library is provided in FIG. 12.
Retained display library members are recovered from the support and
analyzed. The analysis can include amplification and a subsequent
selection under similar or dissimilar conditions. For example,
positive and negative selections can be alternated. The analysis
can also include determining the amino acid sequence of the
polypeptide component and purification of the polypeptide component
for detailed characterization.
Antibodies obtained following a method known in the art and
described herein can be characterized using methods well known in
the art. For example, one method is to identify the epitope to
which the antigen binds, or "epitope mapping." There are many
methods known in the art for mapping and characterizing the
location of epitopes on proteins, including solving the crystal
structure of an antibody-antigen complex, competition assays, gene
fragment expression assays, and synthetic peptide-based assays, as
described, for example, in Chapter 11 of Harlow and Lane, Using
Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y., 1999. In an additional example,
epitope mapping can be used to determine the sequence to which an
antibody binds. The epitope can be a linear epitope, i.e.,
contained in a single stretch of amino acids, or a conformational
epitope formed by a three-dimensional interaction of amino acids
that may not necessarily be contained in a single stretch (primary
structure linear sequence). Peptides of varying lengths (e.g., at
least 4-6 amino acids long) can be isolated or synthesized (e.g.,
recombinantly) and used for binding assays with an antibody. In
another example, the epitope to which the antibody binds can be
determined in a systematic screening by using overlapping peptides
derived from the target antigen sequence and determining binding by
the antibody. According to the gene fragment expression assays, the
open reading frame encoding the target antigen is fragmented either
randomly or by specific genetic constructions and the reactivity of
the expressed fragments of the antigen with the antibody to be
tested is determined. The gene fragments may, for example, be
produced by PCR and then transcribed and translated into protein in
vitro, in the presence of radioactive amino acids. The binding of
the antibody to the radioactively labeled antigen fragments is then
determined by immunoprecipitation and gel electrophoresis.
Certain epitopes can also be identified by using large libraries of
random peptide sequences displayed on the surface of phage
particles (phage libraries). Alternatively, a defined library of
overlapping peptide fragments can be tested for binding to the test
antibody in simple binding assays. In an additional example,
mutagenesis of an antigen binding domain, domain swapping
experiments and alanine scanning mutagenesis can be performed to
identify residues required, sufficient, and/or necessary for
epitope binding. For example, domain swapping experiments can be
performed using a mutant of a target antigen in which various
fragments of the HMWK polypeptide have been replaced (swapped) with
sequences from a closely related, but antigenically distinct
protein. By assessing binding of the antibody to the mutant HMWK,
the importance of the particular antigen fragment to antibody
binding can be assessed.
Alternatively, competition assays can be performed using other
antibodies known to bind to the same antigen to determine whether
an antibody binds to the same epitope as the other antibodies.
Competition assays are well known to those of skill in the art.
Any of the anti-cleaved HMWK antibodies is also within the scope of
the present disclosure.
(iii) Immunoassays
Provided herein are immunoassays for detecting a cleaved HMWK. As
used herein, the term "immunoassay" may be referred to
interchangeably as an immune-based assay or immuno-based assay. In
general, an immunoassay detects the presence and/or concentration
(level) of a molecule (e.g., HMWK), in a sample using an agent that
binds to the molecule, such as an antibody. Examples of
immunoassays include Western blots, enzyme linked immunosorbent
assays (ELISAs), lateral flow assay, radioimmunoassays,
electrochemiluminescence-based detection assays, magnetic
immunoassays, and related techniques. In some embodiments, the
immunoassay is an ELISA assay. In some embodiments, the immunoassay
is a sandwich ELISA assay. In some embodiments, the immunoassay is
a lateral flow assay.
ELISAs are known in the art (see, e.g., Crowther, John R (2009).
"The ELISA Guidebook." 2nd ed. Humana Press and Lequin R (2005).
"Enzyme immunoassay (EIA)/enzyme-linked immunosorbent assay
(ELISA)". Clin. Chem. 51 (12): 2415-8) and exemplary ELISAs are
described herein. Kits for performing ELISAs are also known in the
art and commercially available (see, e.g., ELISA kits from Life
Technologies and BD Biosciences).
To perform the immunoassay described herein, a sample may be
obtained from a subject. As used herein, a "sample" refers to a
composition that comprises tissue, e.g., blood, plasma or protein,
from a subject. A sample includes both an initial unprocessed
sample taken from a subject as well as subsequently processed,
e.g., partially purified or preserved forms. Exemplary samples
include blood, plasma, tears, or mucus. In some embodiments, the
sample is a body fluid sample such as a serum or plasma sample. A
sample to be analyzed by the immunoassay described herein can be
either an initial unprocessed sample taken from a subject or
subsequently processed, e.g., partially purified or preserved
forms. In some embodiments, multiple (e.g., at least 2, 3, 4, 5, or
more) samples may be collected from the subject, over time or at
particular time intervals, for example to assess the progression of
a disease or disorder or evaluate the efficacy of a treatment. The
multiple samples may be obtained before and after a treatment, or
during the course of a treatment.
A sample can be obtained from a subject using any means known in
the art. In some embodiments, the sample is obtained from the
subject by collecting the sample (e.g., a blood sample) into an
evacuated collection tube (e.g., an evacuated blood collection
tube). In some embodiments, the evacuated collection tube contains
one or more protease inhibitors, for example, to reduce or prevent
ex vivo activation of the contact system during sample collection.
Such protease inhibitors may be contained in a liquid formulation.
In some embodiments, the protease inhibitors comprise at least one
serine protease inhibitor and at least one cysteine protease
inhibitor. Such evacuated collection tubes are known in the art.
See, for example, PCT Application No. US2016/046681. Optionally, an
evacuated blood collection tube may further comprise one or more
anti-coagulants.
A "patient," "subject" or "host" (these terms are used
interchangeably) to be treated by the subject method may mean
either a human or non-human animal. In some embodiments, a subject
is suspected of or is at risk for or suffers from a
kallikrein-mediated disorder, e.g., a bradykinin-mediated disorder,
such as hereditary angioedema (HAE), non-histamine-dependent
idiopathic angioedema, rheumatoid arthritis, Crohn's disease,
lupus, Alzheimer's disease, septic shock, burn injury, brain
ischemia/reperfusion injury, cerebral edema, diabetic retinopathy,
diabetic nephropathy, macular edema, vasculitis, arterial or venous
thrombosis, thrombosis associated with ventricular assist devices
or stents, heparin-induced thrombocytopenia with thrombosis,
thromboembolic disease, and coronary heart disease with unstable
angina pectoris, edema, eye disease, gout, intestinal bowel
disease, oral mucositis, neuropathic pain, inflammatory pain,
spinal stenosis-degenerative spine disease, post operative ileus,
aortic aneurysm, osteoarthritis, hereditary angioedema, pulmonary
embolism, stroke, head trauma or peri-tumor brain edema, sepsis,
acute middle cerebral artery (MCA) ischemic event (stroke),
restenosis (e.g., after angioplasty), systemic lupus erythematosis
nephritis, an autoimmune disease, an inflammatory disease, a
cardiovascular disease, a neurological disease, a disease
associated with protein misfolding, a disease associated with
angiogenesis, hypertensive nephropathy and diabetic nephropathy,
allergic and respiratory diseases (e.g., anaphylaxis, asthma,
chronic obstructive pulmonary disease, acute respiratory distress
syndrome, cystic fibrosis, persistent, rhinitis) and tissue
injuries (e.g., burn or chemical injury).
Alternatively or in addition, the subject who needs the analysis
described herein may be a patient of the disease or disorder. Such
a subject may be under the attack of the disease (e.g., HAE)
currently, or may suffer from the disease in the past (e.g., during
disease quiescence currently). In some examples, the subject is a
human patient who may be on a treatment of the disease, for
example, a treatment involving a C1 esterase inhibitor (C1-INH), a
plasma kallikrein inhibitor, or a bradykinin inhibitor. In other
instances, such a human patient may be free of such a
treatment.
The sample described herein can be subject to analysis using an
agent that specifically binds a cleaved HMWK to determine the level
of the cleaved HMWK in the sample. In some embodiments, the
immunoassays described herein may in the format of a sandwich
ELISA, in which a first agent (e.g., the antibody described herein)
that specifically binds the cleaved HMWK is immobilized on a
support member. The support member can then be incubated with a
sample as described herein for a suitable period of time under
conditions that allow for the formation of cleaved HMWK/first agent
(e.g., antibody) complex. Such a complex can then be detected using
a second agent that binds HMWK. The second agent can be conjugated
to a label, which can release a signal directly or indirectly. The
intensity of the signal represents the level of the cleaved HMWK in
the sample.
Any support member known in the art may be used in the method,
including but not limited to a membrane, a bead, a slide, or a
multi-well plate. Selection of an appropriate support member for
the immunoassay will depend on various factor such as the number of
samples and method of detecting the signal released from label
conjugated to the second agent.
In some embodiments, the support member is a membrane, such as a
nitrocellulose membrane, a polyvinylidene fluoride (PVDF) membrane,
or a cellulose acetate membrane. In some examples, the immunoassay
may be in a Western blot assay format or a lateral flow assay
format.
In some embodiments, the support member is a multi-well plate, such
as an ELISA plate. In some embodiments, the immunoassays described
herein can be carried out on high throughput platforms. In some
embodiments, multi-well plates, e.g., 24-, 48-, 96-, 384- or
greater well plates, may be used for high throughput immunoassays.
Individual immunoassays can be carried out in each well in
parallel. Therefore, it is generally desirable to use a plate
reader to measure multiple wells in parallel to increase assay
throughput. In some embodiments, plate readers that are capable of
imaging multi-wells (e.g., 4, 16, 24, 48, 96, 384, or greater
wells) in parallel can be used for this platform. For example, a
commercially available plate reader (e.g., the plate::vision system
available from Perkin Elmer, Waltham, Mass.) may be used. This
plate reader is capable of kinetic-based fluorescence analysis. The
plate::vision system has high collection efficiency optics and has
special optics designed for the analysis of 96 wells in parallel.
Additional suitable parallel plate readers include but are not
limited to the SAFIRE (Tecan, San Jose, Calif.), the
FLIPRTETRA.RTM. (Molecular Devices, Union City, Calif.), the
FDSS7000 (Hamamatsu, Bridgewater, N.J.), and the CellLux (Perkin
Elmer, Waltham, Mass.).
As described in Example 1, it was unexpectedly discovered that the
surface area and/or volume of the wells of the multi-well plate may
affect the results of the immunoassay. In some embodiments, the
described immunoassays are performed in 96-well plates, such as a
96-well ELISA plate.
In other embodiments, high-throughput screening immunoassays of the
present disclosure can be automated (e.g., adapted to robotic
assays).
In some embodiments, the immunoassays may be performed on
low-throughput platforms, including single immunoassay format. For
example, a low-throughput platform may be used to measure the
presence and amount of cleaved HMWK in biological samples (e.g.,
biological tissues, tissue extracts) for diagnostic methods,
monitoring of disease and/or treatment progression, and/or
predicting whether a disease or disorder may benefit from a
particular treatment.
Any method known in the art can be used to immobilize an agent that
specifically binds a cleaved HMWK such as the antibodies described
herein onto a support member as also described herein. In some
embodiments, the immobilization involves binding the agent (e.g.,
the antibody) to the support member. In other embodiments, the
immobilization involves adsorbing the antibody to the support
member. Such adsorption methods may be performed, for example, by
incubating the antibody in a buffer in the wells of a multi-well
plate. In some embodiments, the agent such as the antibody is
provided in a coating buffer and incubated in the wells of a
multi-well plate. Coating buffers will be evident to one of skill
in the art and may be prepared or obtained from a commercial
source. Non-limiting examples of coating buffers include 50 mM
sodium bicarbonate, pH 9.6; 0.2 M sodium bicarbonate, pH 9.4;
phosphate buffered solution (50 mM phosphate, pH 8.0, 0.15 M NaCl);
carbonate-bicarbonate solution; and TBS (50 mM TRIS, pH 8.0, 0.15 M
NaCl).
In some embodiments, the first agent is immobilized on the support
member by hydrophobic interactions between the first agent and the
support member. In some embodiments, the first agent is immobilized
on the support member using electrophoretic transfer.
Either before or after immobilization, or both, the support member
may be incubated with a blocking buffer. In general, blocking
buffers are used to block any of the exposed surface of the support
membrane (e.g., sites on the support membrane unoccupied by the
first agent). Use of a blocking buffer may reduce the baseline
signal detected (i.e., "background interference") and/or improve
the sensitivity of the immunoassay and/or reduce non-specific
binding of components of the sample to the support membrane. As
described in Example 1, selection of the blocking buffer affected
the results of the immunoassay. In some embodiments, the blocking
buffer contains serum albumin, such as bovine serum albumin or
human serum albumin. In some embodiments, the blocking buffer is a
BSA buffer (e.g., 2% BSA in PBS buffer). In some embodiments, the
blocking buffer is free from serum albumin, such as bovine serum
albumin or human serum albumin. In some embodiments, the blocking
buffer comprises casein fragments, and optionally NaCl and Tween
and may have a pH 7.0-7.4. In some embodiments, the casein
fragments are high purity casein fragments. Such a blocking buffer
may be prepared or obtained from a commercial source (e.g., The
Blocking Solution LowCross from CANDOR Bioscience).
The support member, on which the agent specific to a cleaved HMWK
is attached, can be brought in contact (incubated) with a sample as
described herein, which is suspected of containing the cleaved
HMWK. In general, the term "contact" refers to an exposure of the
support member with the biological sample or agent for a suitable
period sufficient for the formation of complexes between the agent,
such as an antibody, and the cleaved HMWK in the sample, if any.
Afterwards, the sample may be removed from the support member,
which can then be washed for multiple times to remove any unbound
cleaved HMWK. In some embodiments, the contacting is performed by
capillary action in which a biological sample or agent is moved
across a surface of the support membrane.
The support member can then be incubated with a second agent that
binds HMWK for a suitable period allowing for the binding of the
second agent to HMWK attached to the support member.
The second agent can be any agent capable of binding to HMWK, such
as an antibody capable of binding to HMWK (either specific to the
cleaved form of HMWK or can cross react to both the cleaved HMWK
and intact HMWK). In some embodiments, the second agent comprises
one or more antibodies that bind HWMK (cleaved and/or intact). In
some embodiments, the antibody is a mouse monoclonal antibody or a
monoclonal sheep antibody. It is conjugated with a label, which is
a compound capable of releasing a signal either directly or
indirectly (e.g., via interaction with one or more additional
compounds).
In some embodiments, the label is a signal releasing agent, which
is an agent that either directly releases a signal (e.g., a dye or
fluorophore) or releases a signal upon interacting with a substrate
(e.g., an enzyme such as HRP or .beta.-galactosidase, which can
convert a colorless substrate to a colored product). As used
herein, the term "fluorophore" (also referred to as "fluorescent
label" or "fluorescent dye") refers to moieties that absorb light
energy at a defined excitation wavelength and emit light energy at
a different wavelength.
In other embodiments, the label can be a member of a
receptor-ligand pair. As used herein, a "ligand-receptor pair"
refers to a pair of molecules (e.g., biological molecules) that
have a specific affinity for each other, e.g., biotin-streptavidin.
In this case, the support member carrying the first agent-cleaved
HMWK-second agent may be further incubated with the other member of
the ligand-receptor pair for a suitable period such that the two
members of the receptor-ligand pair interact. The other member of
the receptor-ligand pair is conjugated with a signal releasing
agent as described herein. In one example, the second agent is
conjugated to biotin and HRP-conjugated streptavidin is used for
detection.
After washing away any unbound conjugate, a substrate solution may
be added to aid in detection. For example, after a set interval,
the reaction may be stopped (e.g., by adding 1 N NaOH) and the
concentration of colored product formed may be measured in a
spectrophotometer. The intensity of color is proportional to the
concentration of bound antigen.
Next, the signal released from the label as described herein can be
detected/measured by routine methodology, which would depend on the
specific format of an immunoassay and the signal releasing agent
used therein. As used herein, the terms "measuring" or
"measurement," or alternatively "detecting" or "detection," means
assessing the presence, absence, quantity or amount (which can be
an effective amount) of a substance within a sample, including the
derivation of qualitative or quantitative concentration levels of
such substances, or otherwise evaluating the values or
categorization of a subject.
Assays, e.g., Western blot assays, may further involve use of a
quantitative imaging system, e.g., LICOR imaging technology, which
is commercially available (see, e.g., the Odyssey.RTM. CLx infrared
imaging system from LI-COR Biosciences). In some embodiments, an
electrochemiluminescence detection assay or an assay relying on a
combination of electrochemiluminescence and patterned array
technology is used (e.g., an ECL or MULTI-ARRAY technology assay
from Meso Scale Discovery (MSD)).
Any of the immunoassays described herein, e.g., one or more steps
of the immunoassays, may be carried out in a suitable assay buffer,
which will be evident to one of skill in the art. In some
embodiments, the assay buffer contains or has been supplemented
with ZnCl.sub.2. In some embodiments, the assay buffer contains at
least about 10 .mu.M, 20 .mu.M, 30 .mu.M, 40 .mu.M, 50 .mu.M, 60
.mu.M, 70 .mu.M, 80 .mu.M, 90 .mu.M, 100 .mu.M, 150 .mu.M, 200
.mu.M, 250 .mu.M, 300 .mu.M, 350 .mu.M, 400 .mu.M, 450 .mu.M, 500
.mu.M or more ZnCl.sub.2. In some embodiments, such a
ZnCl.sub.2-containing assay buffer is used in the step in which the
agent specific to cleaved HMWK (e.g., an antibody specific to
cleaved HMWK) binds a cleaved HMWK. ZnCl.sub.2 enhances the binding
activity of the agent (e.g., antibody) to the cleaved HMWK.
In some embodiments, the assay buffer contains serum albumin, such
as bovine serum albumin or human serum albumin. In some
embodiments, the assay buffer contains at least about 0.01%. 0.02%,
0.03%, 0.04%. 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.12%,
0.014%, 0.16%, 0.18%, 0.2%, 0.25%, 0.3%, 0.4%, or more BSA. In some
embodiments, the assay buffer contains a surfactant, such as
Tween-20. In some embodiments, the assay buffer contains 0.01%.
0.02%, 0.03%, 0.04%. 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1% or
more of a surfactant. In one example, the assay buffer contains
0.1% BSA and 0.05% Tween-20 in PBS.
(iv) Diagnostic and Prognostic Applications
The assay methods and kits described herein can be applied for
evaluation of a disease or disorder associated with plasma
kallikrein, such as those described herein (e.g., HAE), given the
correlation between the level of cleaved HMWK and such diseases or
disorders (e.g. as a biomarker). Alternatively or in addition, the
assay methods and kits described herein may be used to monitor the
progress of such a disease, assess the efficacy of a treatment for
the disease, identify patients suitable for a particular treatment,
and/or predict disease status (e.g., attack versus quiescence) in a
subject.
In some embodiments, the level of cleaved HMWK determined by the
immunoassay described herein can be relied on to evaluate whether a
subject (e.g., a human patient) from whom the biological sample is
obtained, has or is at risk for a disease or disorder associated
with plasma kallikrein, such as HAE or autoimmune disease such as
RA, UC, and Crohn's disease). The level of cleaved kininogen can
then be compared with either the intact kininogen or the total
amount of kininogen in the sample to determine a value (e.g.,
percentage) of cleaved kininogen, a value of intact kininogen, or
both, in the sample. The value of cleaved kininogen and/or intact
kininogen can be compared to a reference value to determine whether
the subject has or is at risk for the PKal-mediated disorder, e.g.,
HAE or an autoimmune disease, such as RA, UC, and Crohn's disease.
For example, if the percentage of cleaved kininogen is at or higher
than a reference number, the subject can be identified as having or
at risk for a pKal-mediated disorder such as HAE, RA, UC, and
Crohn's disease. Alternatively, if the percentage of intact
kininogen is at or lower than a reference number, the subject can
be identified as having or at risk for a pKal-mediated disorder
such as HAE, RA, UC, and Crohn's disease.
In some embodiments, the sample for analysis of the methods
described herein is derived from a human subject who has or is at
risk of having hereditary angioedema (HAE). HAE is also known as
"Quincke edema," C1 esterase inhibitor deficiency, C1 inhibitor
deficiency, and hereditary angioneurotic edema (HANE). HAE is
characterized by recurrent episodes of severe swelling
(angioedema), which can affect, e.g., the limbs, face, genitals,
gastrointestinal tract, and airway. Symptoms of HAE include, e.g.,
swelling in the arms, legs, lips, eyes, tongue, and/or throat;
airway blockage that can involve throat swelling and sudden
hoarseness; repeat episodes of abdominal cramping without obvious
cause; and/or swelling of the intestines, which can be severe and
can lead to abdominal cramping, vomiting, dehydration, diarrhea,
pain, and/or shock. About one-third of individuals with this HAE
develop a non-itchy rash called erythema marginatum during an
attack.
Swelling of the airway can be life threatening and causes death in
some patients. Mortality rates are estimated at 15-33%. HAE leads
to about 15,000-30,000 emergency department visits per year.
Trauma or stress, e.g., dental procedures, sickness (e.g., viral
illnesses such as colds and the flu), menstruation, and surgery can
trigger an attack of angioedema. To prevent acute attacks of HAE,
patients can attempt to avoid specific stimuli that have previously
caused attacks. However, in many cases, an attack occurs without a
known trigger. Typically, HAE symptoms first appear in childhood
and worsen during puberty. On average, untreated individuals have
an attack every 1 to 2 weeks, and most episodes last for about 3 to
4 days (ghr.nlm.nih.gov/condition/hereditary-angioedema). The
frequency and duration of attacks vary greatly among people with
hereditary angioedema, even among people in the same family.
There are three types of HAE, known as types I, II, and III. It is
estimated that HAE affects 1 in 50,000 people, that type I accounts
for about 85 percent of cases, type II accounts for about 15
percent of cases, and type III is very rare. Type III is the most
newly described form and was originally thought to occur only in
women, but families with affected males have been identified.
HAE is inherited in an autosomal dominant pattern, such that an
affected person can inherit the mutation from one affected parent.
New mutations in the gene can also occur, and thus HAE can also
occur in people with no history of the disorder in their family. It
is estimated that 20-25% of cases result from a new spontaneous
mutation.
Mutations in the SERPING1 gene cause hereditary angioedema type I
and type II. The SERPING1 gene provides instructions for making the
C1 inhibitor protein, which is important for controlling
inflammation. C1 inhibitor blocks the activity of certain proteins
that promote inflammation. Mutations that cause hereditary
angioedema type I lead to reduced levels of C1 inhibitor in the
blood. In contrast, mutations that cause type II result in the
production of a C1 inhibitor that functions abnormally. Without the
proper levels of functional C1 inhibitor, excessive amounts of
bradykinin are generated. Bradykinin promotes inflammation by
increasing the leakage of fluid through the walls of blood vessels
into body tissues. Excessive accumulation of fluids in body tissues
causes the episodes of swelling seen in individuals with hereditary
angioedema type I and type II.
Mutations in the F12 gene are associated with some cases of
hereditary angioedema type III. The F12 gene provides instructions
for making coagulation factor XII. In addition to playing a
critical role in blood clotting (coagulation), factor XII is also
an important stimulator of inflammation and is involved in the
production of bradykinin. Certain mutations in the F12 gene result
in the production of factor XII with increased activity. As a
result, more bradykinin is generated and blood vessel walls become
more leaky, which leads to episodes of swelling. The cause of other
cases of hereditary angioedema type III remains unknown. Mutations
in one or more as-yet unidentified genes may be responsible for the
disorder in these cases.
HAE can present similarly to other forms of angioedema resulting
from allergies or other medical conditions, but it differs
significantly in cause and treatment. When HAE is misdiagnosed as
an allergy, it is most commonly treated with antihistamines,
steroids, and/or epinephrine, which are typically ineffective in
HAE, although epinephrine can be used for life-threatening
reactions. Misdiagnoses have also resulted in unnecessary
exploratory surgery for patients with abdominal swelling, and in
some HAE patients abdominal pain has been incorrectly diagnosed as
psychosomatic.
C1 inhibitor therapies, as well as other therapies for HAE, are
described in Kaplan, A. P., J Allergy Clin Immunol, 2010,
126(5):918-925.
Acute treatment of HAE attacks is provided to halt progression of
the edema as quickly as possible. C1 inhibitor concentrate from
donor blood, which is administered intravenously, is one acute
treatment; however, this treatment is not available in many
countries. In emergency situations where C1 inhibitor concentrate
is not available, fresh frozen plasma (FFP) can be used as an
alternative, as it also contains C1 inhibitor.
Purified C1 inhibitor, derived from human blood, has been used in
Europe since 1979. Several C1 inhibitor treatments are now
available in the U.S. and two C1 inhibitor products are now
available in Canada. Berinert P (CSL Behring), which is
pasteurized, was approved by the F.D.A. in 2009 for acute attacks.
CINRYZE.RTM., which is nanofiltered, was approved by the F.D.A. in
2008 for prophylaxis. Rhucin/Ruconest (Pharming) is a recombinant
C1 inhibitor under development that does not carry the risk of
infectious disease transmission due to human blood-borne
pathogens.
Treatment of an acute HAE attack also can include medications for
pain relief and/or IV fluids.
Other treatment modalities can stimulate the synthesis of C1
inhibitor, or reduce C1 inhibitor consumption. Androgen
medications, such as danazol, can reduce the frequency and severity
of attacks by stimulating production of C1 inhibitor.
Helicobacter pylori can trigger abdominal attacks. Antibiotics to
treat H. pylori will decrease abdominal attacks.
Newer treatments attack the contact cascade. Ecallantide
(KALBITOR.RTM.) inhibits plasma kallikrein and has been approved in
the U.S. Icatibant (FIRAZYR.RTM., Shire) inhibits the bradykinin B2
receptor, and has been approved in Europe and the U.S.
Diagnosis of HAE can rely on, e.g., family history and/or blood
tests. Laboratory findings associated with HAE types I, II, and III
are described, e.g., in Kaplan, A. P., J Allergy Clin Immunol,
2010, 126(5):918-925. In type I HAE, the level of C1 inhibitor is
decreased, as is the level of C4, whereas C1q level is normal. In
type II HAE, the level of C1 inhibitor is normal or increased;
however, C1 inhibitor function is abnormal. C4 level is decreased
and C1q level is normal. In type III, the levels of C1 inhibitor,
C4, and C1q can all be normal. The present disclosure is based, at
least in part, on the identification of additional proteins that
have differential levels in samples from HAE patients as compared
to healthy individuals (Table 1). Measuring the level or presence
of 2-HMWK can be used to identify whether a subject has a disease,
such as HAE. In some embodiments, the methods may be used to
determine whether a patient has had or is having an HAE attack.
Symptoms of HAE can be assessed, for example, using questionnaires,
e.g., questionnaires that are completed by patients, clinicians, or
family members. Such questionnaires are known in the art and
include, for example, visual analog scales. See, e.g., McMillan, C.
V. et al. Patient. 2012; 5(2):113-26.
The value of cleaved kininogen and/or intact kininogen detected in
a sample from a subject can be compared to a reference value to
determine whether the subject has or is at risk for the
PKal-mediated disorder (e.g., HAE). Alternatively or in addition,
the level of the cleaved kininogen and/or intact kininogen detected
in a sample from the subject can be compared to a reference value
to assess the efficacy of a treatment for the disorder, the
prognosis or severity of the disorder, and/or identifying a subject
as a candidate for treatment.
The reference value can be a control level of cleaved kininogen
percentage. In some embodiments, the control level is the
percentage of cleaved kininogen in a control sample, such as a
sample (e.g., blood or plasma sample) obtained from a healthy
subject or population of healthy subjects, which preferably are of
the same species as the candidate subject. As used herein, a
healthy subject is a subject that is apparently free of the target
disease (e.g., a PKal-mediated disorder such as HAE or autoimmune
diseases such as RA, US, and Crohn's disease) at the time the level
of cleaved and/or intact kininogen is measured or has no history of
the disease.
The control level can also be a predetermined level or threshold.
Such a predetermined level can represent the percentage of cleaved
kininogen in a population of subjects that do not have or are not
at risk for the target disease. It can also represent the
percentage of cleaved kininogen in a population of subjects that
have the target disease.
The predetermined level can take a variety of forms. For example,
it can be single cut-off value, such as a median or mean. In some
embodiments, such a predetermined level can be established based
upon comparative groups, such as where one defined group is known
to have a target disease and another defined group is known to not
have the target disease. Alternatively, the predetermined level can
be a range, for example, a range representing the percentages of
cleaved kininogen in a control population within a predetermined
percentile.
The control level as described herein can be determined by routine
technology. In some examples, the control level can be obtained by
performing a conventional method (e.g., the same assay for
obtaining the level of cleaved and/or intact kininogen in a test
sample as described herein) on a control sample as also described
herein. In other examples, levels of cleaved and/or intact
kininogen can be obtained from members of a control population and
the results can be analyzed by, e.g., a computational program, to
obtain the control level (a predetermined level) that represents
the level of cleaved and/or intact kininogen in the control
population.
By comparing the percentage of cleaved kininogen in a sample
obtained from a candidate subject to the reference value as
described herein, it can be determined as to whether the candidate
subject has or is at risk for the PKal-mediated disease (e.g., HAE
or an autoimmune disease such as RA, UC, and Crohn's disease). For
example, if the percentage of cleaved kininogen in a sample of the
candidate subject deviates from the reference value (e.g.,
increased as compared to the reference value or decreased as
compared to the reference value), the candidate subject might be
identified as having or at risk for the disease. When the reference
value represents represent the percentage range of cleaved
kininogen in a population of subjects that have the target disease,
the percentage of cleaved kininogen in a sample of a candidate
falling in the range indicates that the candidate subject has or is
at risk for the target disease. In some instances, a reference
value may represent a background level indicating absence of
cleaved kininogen. Presence of cleaved kininogen is deemed as a
deviation from such a background reference value. As used herein, a
"deviation from" a control sample or reference value encompasses
levels of cleaved HMWK as well as the presence or absence of
cleaved HMWK in the sample.
As used herein, "an elevated level or a level above a reference
value" means that the level/percentage of cleaved kininogen is
higher than a reference value, such as a pre-determined threshold
of a level/percentage of cleaved kininogen in a control sample.
Control levels are described in detail herein.
An elevated percentage of cleaved kininogen includes a cleaved
kininogen percentag that is, for example, 1%, 5%, 10%, 20%, 30%,
40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, 500% or
more above a reference value. An elevated percentage of cleaved
kininogen also includes increasing a phenomenon from a zero state
(e.g., no or undetectable cleaved kininogen and/or intact kininogen
that binds to a capture reagent in a sample) to a non-zero state
(e.g., some or detectable cleaved kininogen and/or intact
kininogen).
As used herein, "a decreased percentage/level or a percentage/level
below a reference value" means that the percentage/level of cleaved
is lower than a reference value, such as a pre-determined threshold
of cleaved kininogen in a control sample. Control levels are
described in detail herein.
An decreased level of cleaved kininogen includes a cleaved
kininogen that is, for example, 1%, 5%, 10%, 20%, 30%, 40%, 50%,
60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, 500% or more
lower than a reference value. A decreased level of cleaved
kininogen that binds to a capture reagent also includes decreasing
a phenomenon from a non-zero state (e.g., some or detectable
cleaved kininogen in a sample) to a zero state (e.g., no or
undetectable cleaved kininogen in a sample).
In some embodiments, the candidate subject is a human patient
having a symptom of a pKal-mediated disorder, e.g., such as HAE or
an autoimmune disease such as RA, UC, and Crohn's disease. For
example, the subject has edema, swelling wherein said swelling is
completely or predominantly peripheral; hives; redness, pain, and
swelling in the absence of evidence of infection;
non-histamine-mediated edema, recurrent attacks of swelling, or a
combination thereof. In other embodiments, the subject has no
symptom of a pKal-mediated disorder at the time the sample is
collected, has no history of a symptom of a pKal-mediated disorder,
or no history of a pKal-mediated disorder such as HAE. In yet other
embodiments, the subject is resistant to an anti-histamine therapy,
a corticosteroid therapy, or both.
A subject identified in the methods described herein may be subject
to a suitable treatment.
The assay methods and kits described herein can be applied for
evaluation of the efficacy of a treatment for a disease associated
with plasma kallikrein, such as those described herein, given the
correlation between the level of cleaved HMWK and such diseases.
For examples, multiple biological samples (e.g., blood or plasma
samples) can be collected from a subject to whom a treatment is
performed either before and after the treatment or during the
course of the treatment. The levels of cleaved and/or intact
kininogen can be measured by any of the assay methods as described
herein and values (e.g., percentages) of cleaved and/or intact
kininogen can be determined accordingly. If the percentage of the
cleaved kininogen decreases after the treatment or over the course
of the treatment (the cleaved kininogen percentage in a later
collected sample as compared to that in an earlier collected
sample) or the percentage of intact kininogen increases after the
treatment or over the course of the treatment, it indicates that
the treatment is effective. In some examples, the treatment
involves a therapeutic agent, such as a kallikrein inhibitor, a
bradykinin B2 receptor antagonist, or a C1-INH replacement agent.
Examples of the therapeutic agents include, but not limited to,
landadelumab (DX-2930), ecallantide (DX-88), icantibant, and human
plasma-derived C1-INH.
If the subject is identified as not responsive to the treatment, a
higher dose and/or frequency of dosage of the therapeutic agent are
administered to the subject identified. In some embodiments, the
dosage or frequency of dosage of the therapeutic agent is
maintained, lowered, or ceased in a subject identified as
responsive to the treatment or not in need of further treatment.
Alternatively, a different treatment can be applied to the subject
who is found as not responsive to the first treatment.
In other embodiments, the values of cleaved kininogen, either alone
or in combination with that of intact kininogen, can also be relied
on to identify a disorder that may be treatable by a pKal
inhibitor. To practice this method, the level of cleaved kiniogen
and/or the level of intact kininogen in a sample collected from a
subject (e.g., a blood sample or a plasma sample) having a target
disease can be measured by a suitable assay, e.g., those described
herein such as a Western blot or ELISA assay. Values such as
percentages of the cleaved and/or intact kininogen can be
determined as described herein. The values of cleaved kininogen
and/or intact kininogen can be compared with a reference value as
described herein. If the value of cleaved kininogen/intact
kininogen deviates from the reference value (e.g., elevated or
decreased), it indicates that a pKal inhibitor may be effective in
treating the disease. For example, if the percentages of cleaved
kininogen are decreasing after the treatment or over the course of
the treatment, the treatment can be identified as being effective.
Alternatively, if the percentages of intact kininogen are
increasing after the treatment or over the course of the treatment,
the treatment is identified as being effective.
If the disease is identified as being susceptible (can be treated
by) to a pKal inhibitor, the method can further comprise
administering to the subject having the disease an effective amount
of a pKal inhibitor, e.g., ecallantide (DX-88), EPIKAL-2, or
landadelumab (DX-2930).
Also within the scope of the present disclosure are methods of
evaluating the severity of a disease or disorder associated with
plasma kallikrein or the disease state. For example, as described
herein, HAE may be in the quiescent state (basal state), during
which the subject does not experience symptoms of the disease. HAE
attacks are typically recurrent episodes in which the subject may
experience pain and swelling, for example in the hands, feet, face,
gastrointestinal tract, genitals, and larynx (throat) that can last
from two to five days. In some embodiments, the level of 2-HMWK is
indicative of whether the subject will experience, is experiencing,
or will soon experience an HAE attack. In some embodiments, the
methods involve comparing the level of 2-HMWK in a sample obtained
from a subjecting having HAE to the level of 2-HMWK in a sample
from the same subject, for example a sample obtained from the same
subject at basal state or a sample obtained from the same subject
during a HAE attack.
(v) Non-Clinical Applications
Further, assays for detecting the levels of cleaved 2-HMWK
described herein may be used for research purposes. Although many
diseases and disorders associated with or mediated by plasma
kallikrein have been identified, it is possible that other diseases
are mediated by similar mechanisms or involve similar components.
In some embodiments, the methods described herein may be used to
identify a disease as being associated with or mediated by plasma
kallikrein or with components of the contact activation system. In
some embodiments, the methods described herein may be used to study
mechanisms (e.g., the discovery of novel biological pathways or
processes involved in disease development) or progression of a
disease.
In some embodiments, the levels of cleaved 2-HMWK as measured using
the assays described herein, may be relied on in the development of
new therapeutics for a disease associated with the contact
activation system. For example, the levels of cleaved 2-HMWK may be
measured in samples obtained from a subject having been
administered a new therapy (e.g., a clinical trial). In some
embodiments, the levels of cleaved 2-HMWK may indicate the efficacy
of the new therapeutic or the progression of the disease in the
subject prior to, during, or after the new therapy.
II. Treatment of Diseases Associated with Plasma Kallikrein
A subject at risk for or suffering from a disease associated with
plasma kallikrein, as identified using the methods and assays
described herein, may be treated with any appropriate therapeutic
agent. In some embodiments, provided methods include selecting a
treatment for a subject based on the output of the described
method, e.g., measuring the level of cleaved 2-HMWK.
In some embodiments, the method comprises one or both of selecting
or administering a therapeutic agent, e.g., a kallikrein inhibitor,
a bradykinin B2 receptor inhibitor, and/or a C1 esterase inhibitor,
for administration to the subject based on the output of the assay,
e.g., 2-HMWK detection.
In some embodiments, the therapeutic agent is administered one or
more times to the subject. In some embodiments, a plasma kallikrein
inhibitor is administered to a subject. In some embodiments,
kallikrein inhibitor is a peptide, a small molecule inhibitor, a
kallikrein antibody, or a fragment thereof. In some embodiments, an
antagonist of bradykinin B2 receptor is administered to a subject.
In some embodiments, a C1-INH is administered to a subject.
The therapeutic agent, e.g., kallikrein inhibitor, bradykinin B2
receptor inhibitor, and/or C1-INH, may be administered along with
another therapy as part of a combination therapy for treatment of
the disease or condition that involves the contact activation
system. Combination therapy, e.g., with one or more of a kallikrein
inhibitor, bradykinin B2 receptor antagonist, or C1-INH replacement
agent, e.g., with one or more of a kallikrein inhibitor, bradykinin
B2 receptor antagonist or C1-INH replacement agent and another
therapy, may be provided in multiple different configurations. The
first agent may be administered before or after the administration
of the other therapy. In some situations, the first agent and
another therapy (e.g., a therapeutic agent) are administered
concurrently, or in close temporal proximity (e.g., a short time
interval between the injections, such as during the same treatment
session). The first agent and the other therapy may also be
administered at greater temporal intervals.
Plasma kallikrein binding agents (e.g., binding proteins, e.g.,
polypeptides, e.g., inhibitory polypeptides, e.g., antibodies,
e.g., inhibitory antibodies, or other binding agents, e.g., small
molecules) are useful therapeutic agents for a variety of diseases
and conditions, e.g., diseases and conditions that involve plasma
kallikrein activity. For example, in some embodiments, the disease
or condition that involves plasma kallikrein activity is hereditary
angioedema (HAE). In some embodiments a plasma kallikrein binding
agent such as a plasma kallikrein inhibitor is administered to a
subject at risk or suffering from a disease associated with the
contact activation system.
A number of useful protein inhibitors of kallikrein, either tissue
and/or plasma kallikrein, include a Kunitz domain. As used herein,
a "Kunitz domain" is a polypeptide domain having at least 51 amino
acids and containing at least two, and preferably three,
disulfides. The domain is folded such that the first and sixth
cysteines, the second and fourth, and the third and fifth cysteines
form disulfide bonds (e.g., in a Kunitz domain having 58 amino
acids, cysteines can be present at positions corresponding to amino
acids 5, 14, 30, 38, 51, and 55, according to the number of the
BPTI homologous sequences provided below, and disulfides can form
between the cysteines at position 5 and 55, 14 and 38, and 30 and
51), or, if two disulfides are present, they can form between a
corresponding subset of cysteines thereof. The spacing between
respective cysteines can be within 7, 5, 4, 3, 2, 1 or 0 amino
acids of the following spacing between positions corresponding to:
5 to 55, 14 to 38, and 30 to 51, according to the numbering of the
BPTI sequence provided below. The BPTI sequence can be used as a
reference to refer to specific positions in any generic Kunitz
domain. Comparison of a Kunitz domain of interest to BPTI can be
performed by identifying the best fit alignment in which the number
of aligned cysteines in maximized.
The 3D structure (at high resolution) of the Kunitz domain of BPTI
is known. One of the X-ray structures is deposited in the
Brookhaven Protein Data Bank as "6PTI". The 3D structure of some
BPTI homologues (Eigenbrot et al., Protein Engineering (1990)
3(7):591-598; Hynes et al., Biochemistry (1990) 29:10018-10022) are
known. At least eighty one Kunitz domain sequences are known. Known
human homologues include three Kunitz domains of LACI also known as
tissue factor pathway inhibitor (TFPI) (Wun et al., J. Biol. Chem.
(1988) 263(13):6001-6004; Girard et al., Nature (1989) 338:518-20;
Novotny et al, J. Biol. Chem. (1989) 264(31):18832-18837) two
Kunitz domains of Inter-.alpha.-Trypsin Inhibitor, APP-I (Kido et
al. J. Biol. Chem. (1988) 263(34):18104-18107), a Kunitz domain
from collagen, three Kunitz domains of TFPI-2 (Sprecher et al.,
PNAS USA (1994) 91:3353-3357), the Kunitz domains of hepatocyte
growth factor activator inhibitor type 1, the Kunitz domains of
Hepatocyte growth factor activator inhibitor type 2, the Kunitz
domains described in U.S. Patent Publication No.: 2004-0152633.
LACI is a human serum phosphoglycoprotein with a molecular weight
of 39 kDa (amino acid sequence in Table 1) containing three Kunitz
domains.
TABLE-US-00004 TABLE 1 Exemplary Natural Kunitz Domains LACI 1
MIYTMKKVHA LWASVCLLLN LAPAPLNAds eedeehtiit (SEQ ID dtelpplklM NO:
78) 51 HSFCAFKADD GPCKAIMKRF FFNIFTRQCE EFIYGGCEGN QNRFESLEEC 101
KKMCTRDnan riikttlqqe kpdfCfleed pgiCrgyitr yfynnqtkqC 151
erfkyggClg nmnnfetlee CkniCedgpn gfqvdnygtq lnavnnsltp 201
qstkvpslfe fhgpswCltp adrglCrane nrfyynsvig kCrpfkysgC 251
ggnennftsk qeClraCkkg fiqriskggl iktkrkrkkci rvkiayeeif 301 vknm
The signal sequence (1-28) is uppercase and underscored LACI-K1
(50-107) is uppercase LACI-K2 (121-178) is underscored LACI-K3
(211-270) is bold BPTI 1 2 3 4 5 (SEQ ID
1234567890123456789012345678901234567890123456789012345678 NO: 79)
RPDFCLEPPYTGPCKARIIRYFYNAKAGLCQTFVYGGCRAKRNNFKSAEDCMRTCGGA
The Kunitz domains above are referred to as LACI-K1 (residues 50 to
107), LACI-K2 (residues 121 to 178), and LACI-K3 (213 to 270). The
cDNA sequence of LACI is reported in Wun et al. (J. Biol. Chem.
(1988) 263(13):6001-6004). Girard et al. (Nature (1989) 338:518-20)
reports mutational studies in which the P1 residues of each of the
three Kunitz domains were altered. LACI-K1 inhibits Factor VIIa
(F.VIIa) when F.VIIa is complexed to tissue factor and LACI-K2
inhibits Factor Xa.
A variety of methods can be used to identify a Kunitz domain from a
sequence database. For example, a known amino acid sequence of a
Kunitz domain, a consensus sequence, or a motif (e.g., the ProSite
Motif) can be searched against the GenBank sequence databases
(National Center for Biotechnology Information, National Institutes
of Health, Bethesda Md.), e.g., using BLAST; against Pfam database
of HMMs (Hidden Markov Models) (e.g., using default parameters for
Pfam searching; against the SMART database; or against the ProDom
database. For example, the Pfam Accession Number PF00014 of Pfam
Release 9 provides numerous Kunitz domains and an HMM for identify
Kunitz domains. A description of the Pfam database can be found in
Sonhammer et al. Proteins (1997) 28(3):405-420 and a detailed
description of HMMs can be found, for example, in Gribskov et al.
Meth. Enzymol. (1990) 183:146-159; Gribskov et al. Proc. Natl.
Acad. Sci. USA (1987) 84:4355-4358; Krogh et al. J. Mol. Biol.
(1994) 235:1501-1531; and Stultz et al. Protein Sci. (1993)
2:305-314. The SMART database (Simple Modular Architecture Research
Tool, EMBL, Heidelberg, Del.) of HMMs as described in Schultz et
al. Proc. Natl. Acad. Sci. USA (1998) 95:5857 and Schultz et al.
Nucl. Acids Res (2000) 28:231. The SMART database contains domains
identified by profiling with the hidden Markov models of the HMMer2
search program (R. Durbin et al. (1998) Biological sequence
analysis: probabilistic models of proteins and nucleic acids.
Cambridge University Press). The database also is annotated and
monitored. The ProDom protein domain database consists of an
automatic compilation of homologous domains (Corpet et al. Nucl.
Acids Res. (1999) 27:263-267). Current versions of ProDom are built
using recursive PSI-BLAST searches (Altschul et al. Nucleic Acids
Res. (1997) 25:3389-3402; Gouzy et al. Computers and Chemistry
(1999) 23:333-340.) of the SWISS-PROT 38 and TREMBL protein
databases. The database automatically generates a consensus
sequence for each domain. Prosite lists the Kunitz domain as a
motif and identifies proteins that include a Kunitz domain. See,
e.g., Falquet et al. Nucleic Acids Res. (2002) 30:235-238.
Kunitz domains interact with target protease using, primarily,
amino acids in two loop regions ("binding loops"). The first loop
region is between about residues corresponding to amino acids 13-20
of BPTI. The second loop region is between about residues
corresponding to amino acids 31-39 of BPTI. An exemplary library of
Kunitz domains varies one or more amino acid positions in the first
and/or second loop regions. Particularly useful positions to vary,
when screening for Kunitz domains that interact with kallikrein or
when selecting for improved affinity variants, include: positions
13, 15, 16, 17, 18, 19, 31, 32, 34, and 39 with respect to the
sequence of BPTI. At least some of these positions are expected to
be in close contact with the target protease. It is also useful to
vary other positions, e.g., positions that are adjacent to the
aforementioned positions in the three-dimensional structure.
The "framework region" of a Kunitz domain is defined as those
residues that are a part of the Kunitz domain, but specifically
excluding residues in the first and second binding loops regions,
i.e., about residues corresponding to amino acids 13-20 of BPTI and
31-39 of BPTI. Conversely, residues that are not in the binding
loop may tolerate a wider range of amino acid substitution (e.g.,
conservative and/or non-conservative substitutions).
In one embodiment, these Kunitz domains are variant forms of the
looped structure including Kunitz domain 1 of human
lipoprotein-associated coagulation inhibitor (LACI) protein. LACI
contains three internal, well-defined, peptide loop structures that
are paradigm Kunitz domains (Girard, T. et al., Nature (1989)
338:518-520). Variants of Kunitz domain 1 of LACI described herein
have been screened, isolated and bind kallikrein with enhanced
affinity and specificity (see, for example, U.S. Pat. Nos.
5,795,865 and 6,057,287). These methods can also be applied to
other Kunitz domain frameworks to obtain other Kunitz domains that
interact with kallikrein, e.g., plasma kallikrein. Useful
modulators of kallikrein function typically bind and/or inhibit
kallikrein, as determined using kallikrein binding and inhibition
assays.
In some aspects, the plasma kallikrein inhibitor binds to the
active form of plasma kallikrein. In some embodiments, the plasma
kallikrein inhibitor, binds to and inhibits plasma kallikrein,
e.g., human plasma kallikrein and/or murine kallikrein. Exemplary
polypeptide plasma kallikrein agents are disclosed in U.S. Pat.
Nos. 5,795,865, 5,994,125, 6,057,287, 6,333,402, 7,628,983, and
8,283,321, 7,064,107, 7,276,480, 7,851,442, 8,124,586, 7,811,991,
and U.S. Publication No. 20110086801, the entire contents of each
of which is incorporated herein by reference. In some embodiments,
the plasma kallikrein inhibitor is an inhibitory polypeptide or
peptide. In some embodiments, the inhibitory peptide is ecallantide
(also referred to as DX-88 or KALBITOR.RTM.; SEQ ID NO:80). In some
embodiments, the kallikrein inhibitor comprises or consists of an
about 58-amino acid sequence of amino acids 3-60 of SEQ ID NO: 80
or the DX-88 polypeptide having the 60-amino acid sequence of SEQ
ID NO: 80.
Glu Ala Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly Pro Cys Arg
Ala Ala His Pro Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu
Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu
Glu Cys Lys Lys Met Cys Thr Arg Asp (SEQ ID NO: 80).
The plasma kallikrein inhibitor can be full-length antibodies
(e.g., an IgG (e.g., an IgG1, IgG2, IgG3, IgG4), IgM, IgA (e.g.,
IgA1, IgA2), IgD, and IgE) or can include only an antigen-binding
fragment (e.g., a Fab, F(ab')2 or scFv fragment). The binding
protein can include two heavy chain immunoglobulins and two light
chain immunoglobulins, or can be a single chain antibody. The
plasma kallikrein inhibitor can be recombinant proteins such as
humanized, CDR grafted, chimeric, deimmunized, or in vitro
generated antibodies, and may optionally include constant regions
derived from human germline immunoglobulin sequences. In one
embodiment, the plasma kallikrein inhibitor is a monoclonal
antibody.
Exemplary plasma kallikrein binding proteins are disclosed in U.S.
Publication No. 20120201756, the entire contents of which are
incorporated herein by reference. In some embodiments, the
kallikrein binding protein is an antibody (e.g., a human antibody)
having the light and/or heavy chains of antibodies selected from
the group consisting of M162-A04, M160-G12, M142-H08, X63-G06,
X101-A01 (also referred to as DX-2922), X81-B01, X67-D03, X67-G04,
X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06,
X115-A03, X115-D01, X115-F02, X124-G01 (also referred to herein as
DX-2930 or lanadelumab), X115-G04, M29-D09, M145-D11, M06-D09 and
M35-G04. In some embodiments, the plasma kallikrein binding protein
competes with or binds the same epitope as M162-A04, M160-G12,
M142-H08, X63-G06, X101-A01 (also referred to herein as DX-2922),
X81-B01, X67-D03, X67-G04, X81-B01, X67-D03, X67-G04, X115-B07,
X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02,
X124-G01, X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04. In some
embodiments, the plasma kallikrein binding protein is lanadelumab.
See US 20110200611 and US 20120201756, which are incorporated by
reference herein.
An example of a plasma kallikrein inhibitory antibody is
lanadelumab. The amino acid sequences of the heavy chain and light
chain variable regions of lanadelumab are provided below with the
CDR regions identified in boldface and underlined.
TABLE-US-00005 Lanadelumab heavy chain variable region sequence
(SEQ ID NO: 81) EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYIMMWVRQA
PGKGLEWVSG IYSSGGITVY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR
IGVPRRDEFD IWGQGTMVTV SS Lanadelumab light chain variable region
sequence (SEQ ID NO: 82) DIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK
PGKAPKLLIY KASTLESGVP SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ
GTKVEI
In some embodiments, a plasma kallikrein inhibitor can have about
85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher
sequence identity to a plasma kallikrein inhibitor described
herein. In some embodiments, a plasma kallikrein inhibitor can have
about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or
higher sequence identity in the HC and/or LC framework regions
(e.g., HC and/or LC FR 1, 2, 3, and/or 4) to a plasma kallikrein
inhibitor described herein. In some embodiments, a plasma
kallikrein inhibitor can have about 85%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99% or higher sequence identity in the HC
and/or LC CDRs (e.g., HC and/or LC CDR1, 2, and/or 3) to a plasma
kallikrein inhibitor described herein. In some embodiments, a
plasma kallikrein inhibitor can have about 85%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity in the
constant region (e.g., CH1, CH2, CH3, and/or CL1) to a plasma
kallikrein inhibitor described herein.
In some aspects, a small molecule binds and inhibits the active
form of plasma kallikrein.
Bradykinin B2 Receptor Inhibitors
In some embodiments, a bradykinin B2 receptor inhibitor (e.g.,
antagonist) is administered to a subject. Exemplary bradykinin B2
receptor antagonists include icatibant (Firazyr.RTM.), which is a
peptidomimetic drug containing 10 amino acids which block binding
of native bradykinin to the bradykinin B2 receptor.
C1-INH Replacement Agents
In some embodiment, a C1 esterase inhibitor (C1-INH), such as a
replacement C1-INH agent is administered to a subject. Exemplary
C1-INH replacement agents are publicly available and include, for
example, human plasma-derived C1-INH, e.g. Berinert.RTM. and
CINRYZE.RTM..
III. Kits for Detection of Cleaved HMWK
The present disclosure also provides kits for use in evaluating
cleaved HMWK in samples suspected of containing a cleaved HWMK,
e.g., biological samples from human patients. Such kits can
comprise a first agent that specifically binds to cleaved HMWK as
compared to intact HMWK or LMWK. In some embodiments, the first
agent is an antibody, such as any of the antibodies described
herein that specifically bind cleaved HMWK (e.g., 559B-M004 or
functional variants thereof as described herein). In some
embodiments, the kits further comprise a second agent (e.g., an
antibody binding to HMWK) for detecting binding of the first agent
to the cleaved HMWK. The second agent can be conjugated to a label.
In some embodiments, the second agent is an antibody that
specifically binds cleaved HMWK. In other embodiments, the second
agent is an antibody that cross reacts with both cleaved and intact
HMWK.
The kit may further comprise a support member for performing the
immunoassay and immobilizing the first agent. In some embodiments,
the support member is a 96-well plate, such as a 96-well ELISA
plate. The kit can also comprise one or more buffers as described
herein but not limited to a coating buffer; an assay buffer, such
as an assay buffer containing ZnCl.sub.2; a blocking buffer; a wash
buffer; and/or a stopping buffer.
In some embodiments, the kit can comprise instructions for use in
accordance with any of the methods described herein. The included
instructions can comprise a description of how to use the
components contained in the kit for measuring the level of cleaved
and/or intact HMWK in a sample, which can be a biological sample
collected from a human patient. Alternatively or in addition, the
kit may comprise may comprise a description of how to use
components contained in the kit for measuring the level of
LMWK.
The instructions relating to the use of the kit generally include
information as to the amount of each component and suitable
conditions for performing the assay methods described herein. The
components in the kits may be in unit doses, bulk packages (e.g.,
multi-dose packages), or sub-unit doses. Instructions supplied in
the kits of the present disclosure are typically written
instructions on a label or package insert (e.g., a paper sheet
included in the kit), but machine-readable instructions (e.g.,
instructions carried on a magnetic or optical storage disk) are
also acceptable.
The label or package insert indicates that the kit is used for
evaluating the level of cleaved and/or intact HMWK. In some
embodiments, the kit is used for evaluating the level of LWMK.
Instructions may be provided for practicing any of the methods
described herein.
The kits of this present disclosure are in suitable packaging.
Suitable packaging includes, but is not limited to, vials, bottles,
jars, flexible packaging (e.g., sealed Mylar or plastic bags), and
the like. Also contemplated are packages for use in combination
with a specific device, such as an inhaler, nasal administration
device (e.g., an atomizer) or an infusion device such as a
minipump. A kit may have a sterile access port (for example the
container may be an intravenous solution bag or a vial having a
stopper pierceable by a hypodermic injection needle). The container
may also have a sterile access port (for example the container may
be an intravenous solution bag or a vial having a stopper
pierceable by a hypodermic injection needle).
Kits may optionally provide additional components such as
interpretive information, such as a control and/or standard or
reference sample. Normally, the kit comprises a container and a
label or package insert(s) on or associated with the container. In
some embodiments, the present disclosure provides articles of
manufacture comprising contents of the kits described above.
IV. Other Antibodies Binding to Cleaved HMWK
Also provided herein are isolated antibodies that bind both cleaved
HMWK and intact HMWK. In some embodiments, such antibodies do not
bind LMWK or bind to LMWK with a low affinity. In other
embodiments, such antibodies also bind to LMWK.
In some embodiments, the antibodies that specifically binds a
cleaved HMWK and intact HMWK (or additionally LMWK) described
herein have a suitable binding affinity to one or more of the
target antigens. The antibody described herein may have a binding
affinity (K.sub.D) of at least 10.sup.-5, 10.sup.-6, 10.sup.-7,
10.sup.-8, 10.sup.-9, 10.sup.-10 M, or lower.
Examples of the antibodies noted above and their binding
specificities are provided in Table 2 in Example 2 below. The amino
acid sequences of the heavy chain and light chain variable regions
are provided below with the CDR regions identified in boldface and
underlined (determined by one scheme as an example):
TABLE-US-00006 >559B-R0049-A01 (559B-M0067-E02) Heavy Chain
Amino Acid Sequence (SEQ ID NO: 6)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSLYPMVWVRQAPGKGLEWVSSIYPSGGFTTYADSV
KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSSRYYYYGMDVWGQGTTVTVSS
>559B-R0049-A01 (559B-M0067-E02) Light Chain Amino Acid Sequence
(SEQ ID NO: 7)
QYELTQPPSMSGTPGQRVTISCSGSSSNIGSEYVYWFQQLPGTAPKLLIYRNDQRPSGVPDRFS
GSKSGTSASLAISGLRSEDETDYYCSTWDDTLRTGVFGGGTKVTVL >559B-R0049-G05
(559B-M0039-G07) Heavy Chain Amino Acid Sequence (SEQ ID NO: 8)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYRMRWVRQAPGKGLEWVSGISPSGGWTYYADSV
KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTTDNGDYALAHWGQGTLVTVSS
>559B-R0049-G05 (559B-M0039-G07) Light Chain Amino Acid Sequence
(SEQ ID NO: 9)
QDIQMTQSPSSLSASVGDRVTITCRASQRIINYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFS
GSGSGTDFTLTISSLQPEDFATYYCQQSYSAPLTFGGGTRVEIK >559B-R0048-A09
(559B-M0044-E09) Heavy Chain Amino Acid Sequence (SEQ ID NO: 10)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSQYSMGWVRQAPGKGLEWVSSIYSSGGSTQYADSV
KGRFTISRDNSKNTLYLQMNSLRAEDTATYYCARTFRRGWFGEDYYYYMDVWGKGTTVTVSS
>559B-R0048-A09 (559B-M0044-E09) Light Chain Amino Acid Sequence
(SEQ ID NO: 11)
QDIQMTQSPSSLSASVGDRITITCRASQGIRNDVGWYQQKPGKAPQRLIYAASSLQSGVPSRFS
GSGSGTEFTLTISSLQPEDFATYYCLQHNSYPLTFGGGTKVEIK >559B-R0048-E01
(559B-M0003-008) Heavy Chain Amino Acid Sequence (SEQ ID NO: 12)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSPYMMYWVRQAPGKGLEWVSSISPSGGKTWYADSV
KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARLGGSSSYYYYYYYGMDVWGQGTTVTVSS
>559B-R0048-E01 (559B-M0003-008) Light Chain Amino Acid Sequence
(SEQ ID NO: 13)
QSALTQSPSASGTPGQRVTISCSGSSSNIGGNTVNWYQQFPGTAPKLLIYSNNQRPSGVPDRFS
GSKSGTSASLAISGLQSEDEAIYYCASWDDRLNGHWVFGGGTRLTVL >559B-R0049-G01
(559B-M0039-H06) Heavy Chain Amino Acid Sequence (SEQ ID NO: 14)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSAYDMHWVRQAPGKGLEWVSSIWPSGGGTYYADSV
KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGDYDYGDFTDAFDIWGQGTMVTVSS
>559B-R0049-G01 (559B-M0039-H06) Light Chain Amino Acid Sequence
(SEQ ID NO: 15)
QSALTQPASVSGSPGQSITISCTGTSSDVGSYNLVSWYQQHPGKAPKLMIYEGSKRPSGVPDRF
SGSKSGNTASLIISGLQAEDEADYYCCSYAGSYSYVFGTGTRVTVL >559B-R0049-E05
(559B-M0039-D08) Heavy Chain Amino Acid Sequence (SEQ ID NO: 16)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYAMQWVRQAPGKGLEWVSWIYSSGGPTYYADSV
KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGLPGQPFDYWGQGTLVTVSS
>559B-R0049-E05 (559B-M0039-D08) Light Chain Amino Acid Sequence
(SEQ ID NO: 17)
QSELTQPPSASGTPGQRVTISCSGSSSNIGNNYVYWYQQFPGTAPKLLIYRNNQRPSGVPDRFS
GSKSGTSASLAISGLRSEDEADYYCATWDDRLSGWVFGGGTKLTVL >559B-R0048-A11
(559B-M0068-007) Heavy Chain Amino Acid Sequence (SEQ ID NO: 18)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYQMHWVRQAPGKGLEWVSGIYSSGGSTPYADSV
KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGHHGMDVWGQGT TVTVSS
>559B-R0048-A11 (559B-M0068-007) Light Chain Amino Acid Sequence
(SEQ ID NO: 19)
QDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASNLQSGVPSRFS
GSGSGTDFTLTISSLQPEDFATYYCQKYNIAPYTFGQGTKLEIK >559B-R0048-A03
(559B-M0021-G11) Heavy Chain Amino Acid Sequence (SEQ ID NO: 20)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSPYPMTWVRQAPGKGLEWVSGISSSGGFTPYADSV
KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARMVRGVIKAFDIWGQGTMVTVSS
>559B-R0048-A03 (559B-M0021-G11) Light Chain Amino Acid Sequence
(SEQ ID NO: 21)
QYELTQPPSASGTPGQRVTISCSGSSSNIGSHYVFWYQQLPGAAPKLLIYRNNQRPSGVPDRFS
GSKSGTSASLAISGLRSEDEADYYCATWDNSLSAWVFGGGTKLTVL >559B-R0048-005
(559B-M0061-G06) Heavy Chain Amino Acid Sequence (SEQ ID NO: 22)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSKYTMWWVRQAPGKGLEWVSVISSSGGKTYYADSV
KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTANRAFDIWGQGTMVTVSS
>559B-R0048-005 (559B-M0061-G06) Light Chain Amino Acid Sequence
(SEQ ID NO: 23)
QDIQMTQSPAALSVSPGERATLSCRASQSVSSDLAWYQQKPGQAPRLLIHGASTRATGIPARFS
GSGSGREFTLTISSLQSEDFAVYYCQQYNDWPPLFGPGTKVNIK >559B-R0049-A03
(559B-M0036-G12) Heavy Chain Amino Acid Sequence (SEQ ID NO: 24)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYYMAWVRQAPGKGLEWVSGIVPSGGQTGYADSV
KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTRRGWFGEDYYYYMDVWGKGTLVTVSS
>559B-R0049-A03 (559B-M0036-G12) Light Chain Amino Acid Sequence
(SEQ ID NO: 25)
QDIQMTQSPGILSLSPGERATVSCRASQSVGSTYLAWYQHKPGQAPRLLIYGASSRATGIPDRF
SGSGSGTDFTLTISSLEPEDFAIYYCQHFHTSPPGITFGQGTRLEIK >559B-R0048-009
(559B-M0042-E06) Heavy Chain Amino Acid Sequence (SEQ ID NO: 26)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSMYKMSWVRQAPGKGLEWVSVISPSGGRTYYADSV
KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGTRTSGLDYWGQGTLVTVSS
>559B-R0048-009 (559B-M0042-E06) Light Chain Amino Acid Sequence
(SEQ ID NO: 27)
QSALTQPASVSGSPGQSITISCTGTSSDVGGYKYVSWYQQHPGKAPKLVIYEVSNRPSGVSNRF
SGSKSGNTASLTISGLQAEDEADYYCSSYTSSTTVVFGGGTKLTVL >559B-R0048-E09
(559B-M0070-H10) Heavy Chain Amino Acid Sequence (SEQ ID NO: 28)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYGMRWVRQAPGKGLEWVSVISPSGGKTNYADSV
KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGRPDYYAMDVWGQGTTVTVSS
>559B-R0048-E09 (559B-M0070-H10) Light Chain Amino Acid Sequence
(SEQ ID NO: 29)
QSALTQPPSASGAPGQRVTISCSGSSSNIGSNTVNWYQKLPGTAPKLLIYYNDRRPSGVPDRFS
GSKSGNTASLIISGLQAEDEADYYCAAWDDSLSGPVFGGGTKLTVL >559B-R0048-E05
(559B-M0068-D01) Heavy Chain Amino Acid Sequence (SEQ ID NO: 30)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSIYPMSWVRQAPGKGLEWVSGISPSGGKTAYADSV
KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGQGRAVRGKLYYYGMDVWGQGTTVTVSS
>559B-R0048-E05 (559B-M0068-D01) Light Chain Amino Acid Sequence
(SEQ ID NO: 31)
QSALTQPPSASQTPGQTVTISCSGSSSNIGTNNVNWYQQLPGTAPKLLISSHHRRPSGVPDRFS
ASKSGTSASLAISGLQSEDEADYYCAAWDDSLNGPVFGGGTKLTVL >559B-R0048-001
(559B-M0004-E08) Heavy Chain Amino Acid Sequence (SEQ ID NO: 32)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSMYHMNWVRQAPGKGLEWVSSIYSSGGSTRYADSV
KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGVRYGMDVWGQGTTVTVSS
>559B-R0048-001 (559B-M0004-E08) Light Chain Amino Acid Sequence
(SEQ ID NO: 33)
QDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFS
GSGSGTDFTLTISSLQPEDFATYYCQQANSFPITFGQGTRLEIK >559B-R0049-001
(559B-M0069-009) Heavy Chain Amino Acid Sequence (SEQ ID NO: 34)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSMYDMHWVRQAPGKGLEWVSSISSSGGYTQYADSV
KGRFTISRDNSKNTLYLQMNSLRAEDTAMYYCARDRGLIAAAGGFDPWGQGTLVTVSS
>559B-R0049-001 (559B-M0069-009) Light Chain Amino Acid Sequence
(SEQ ID NO: 35)
QDIQMTQSPSSLSASVGDRVTITCRASQSIGIYLNWYQQKPGTAPKLLIYAASSLQSGVPSRFT
GSGSGTDFTLTISSLQPDDFATYYCQRTYGRPLTFGGGTKVEIK >559B-R0049-A05
(559B-M0038-F04) Heavy Chain Amino Acid Sequence (SEQ ID NO: 36)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSKYEMMWVRQAPGKGLEWVSSISPSGGYTMYADSV
KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHRSKWNDAPFDSWGQGTLVTVSS
>559B-R0049-A05 (559B-M0038-F04) Light Chain Amino Acid Sequence
(SEQ ID NO: 37)
QDIQMTQSPSSLSASVGDRVAITCRASQSIDTYLNWYQQKPGKAPKLLIYAASKLEDGVPSRFS
GSGTGTDFTLTIRSLQPEDFASYFCQQSYSSPGITFGPGTKVEIK >559B-R0048-G05
(559B-M0044-005) Heavy Chain Amino Acid Sequence (SEQ ID NO: 38)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSIYQMYWVRQAPGKGLEWVSSIYSSGGRTFYADSV
KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATRGSWYVGGNEYFQHWGQGTLVTVSS
>559B-R0048-G05 (559B-M0044-005) Light Chain Amino Acid Sequence
(SEQ ID NO: 39)
QSVLTQSPSLSLSPGQTASIPCSGDTLGNKFVSWYQQKPGQSPVLVIYQDTKRPSGIPERFSGS
NSGNTATLTITGTQAMDEADYYCQVWDSNSYAFGPGTKVTVL >559B-R0048-C11
(559B-M0047-H01) Heavy Chain Amino Acid Sequence (SEQ ID NO: 40)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSFYMMYWVRQAPGKGLEWVSSISSSGGFTRYADSV
KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVRGLAVAAPDYWGQGTLVTVSS
>559B-R0048-C11 (559B-M0047-H01) Light Chain Amino Acid Sequence
(SEQ ID NO: 41)
QSELTQPASVSGSPGQSITISCIGTSSDIGTYNYVSWYQQHPGKAPKLMIYDVNTRPSGVSDRF
SGSKSGNTASLTISGLQAEDEADYYCSSYTTSVTWVFGGGTTLTVL >559B-R0048-0O3
(559B-M0019-E12) Heavy Chain Amino Acid Sequence (SEQ ID NO: 42)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSGYNMYWVRQAPGKGLEWVSRISPSGGWTSYADSV
KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRGQWMDWWGQGTMVIVSS
>559B-R0048-0O3 (559B-M0019-E12) Light Chain Amino Acid Sequence
(SEQ ID NO: 43)
QDIQMTQSPSSLSASVGDRVIITCRASQNITGYLNWYQQKPGKAPNLLIYDASRMNTGVPSRFR
GSGSGTDYILTIYKLEPEDIGTYFCQHTDDFSVTFGGGTKVDLK >559B-R0048-A05
(559B-X0004-B05) Heavy Chain Amino Acid Sequence (SEQ ID NO: 44)
EVQLLESGGGLVQPGGSLRLSCAASGFTFHYRMMWVRQAPGKGLEWVSYISSSGGYTAYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAKRNRAFDIWGQGTMVIVSS
>559B-R0048-A05 (559B-X0004-B05) Light Chain Amino Acid Sequence
(SEQ ID NO: 45)
QDIQMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESG
VPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPLGFGQGTKLEIK
>559B-R0048-E11 (559B-M0048-D12) Heavy Chain Amino Acid Sequence
(SEQ ID NO: 46)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYQMTWVRQAPGKGLEWVSSIGSSGGFTNYADSV
KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARLPANFYYYMDVWGKGTTVTVSS
>559B-R0048-E11 (559B-M0048-D12) Light Chain Amino Acid Sequence
(SEQ ID NO: 47)
QDIQMTQSPSSLSASVGDRVTITCRASQNIYSFLNWYQQKPGKAPKLLIYATSSLQSGVPSRFS
GSGSGTDFTLTISSLQPEDFASYYCQQNYNIPWTFGQGTKVEIK >559B-R0048-G11
(559B-M0053-G01) Heavy Chain Amino Acid Sequence (SEQ ID NO: 48)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSWYMMKWVRQAPGKGLEWVSSIVPSGGWTTYADSV
KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATEGNLWFGEGRAFDIWGQGTMVTVSS
>559B-R0048-G11 (559B-M0053-G01) Light Chain Amino Acid Sequence
(SEQ ID NO: 49)
QDIQMTQSPGILSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRF
SGSGSGTDFTLTISRLEPEDFAVYYCQQRSNWPPSFGQGTRLDIK >559B-R0049-005
(559B-M0038-H03) Heavy Chain Amino Acid Sequence (SEQ ID NO: 50)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSKYDMHWVRQAPGKGLEWVSRISSSGGKTEYADSV
KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREYRYCTANTCSLYGMDVWGRGTTVTVSS
>559B-R0049-005 (559B-M0038-H03) Light Chain Amino Acid Sequence
(SEQ ID NO: 51)
QDIQMTQSPSSLSASVGDRVAITCRTSQGVRSDFAWYQQTPGKAPRRLIYAAFILDNGVPSRFS
GSGSGTEFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVEMK >559B-R0048-E03
(559B-M0017-H08) Heavy Chain Amino Acid Sequence (SEQ ID NO: 52)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSPYWMHWVRQAPGKGLEWVSVISPSGGGTGYADSV
KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARESRGSGSHEDYWGQGTLVTVSS
>559B-R0048-E03 (559B-M0017-H08) Light Chain Amino Acid Sequence
(SEQ ID NO: 53)
QDIQMTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYGASNRGTGIPARFS
GSGSGTEFTLTISSLQSEDFAVYFCQQYKNWPNLTFGGGTKVDIK >559B-R0049-E03
(559B-M0035-F05) Heavy Chain Amino Acid Sequence (SEQ ID NO: 54)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYPMAWVRQAPGKGLEWVSGIVSSGGRTVYADSV
KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDPYDFWSEGAFDIWGQGTMVTVSS
>559B-R0049-E03 (559B-M0035-F05) Light Chain Amino Acid Sequence
(SEQ ID NO: 55)
QSVLTQPPSASGTPGQRVTISCSGSSSNIGNNFVYWYHQVPGTAPKLLIYKNNQRPSGVPDRFS
GSKSAASASLAISGLRSEDEADYYCAAWDNSLSGFYVFGAGTKVTVL >559B-R0049-G03
(559B-M0035-H09) Heavy Chain Amino Acid Sequence (SEQ ID NO: 56)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSWYGMHWVRQAPGKGLEWVSRIGPSGGPTSYADSV
KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGYYGTGRYFQHWGQGTLVTVSS
>559B-R0049-G03 (559B-M0035-H09) Light Chain Amino Acid Sequence
(SEQ ID NO: 57)
QDIQMTQSPDSLSLSPGDRATLSCRASQSVGSDYLAWYQQKPGQAPRLLIYDASNRATGIPARF
SGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGGGTKVEIK >559B-R0048-A07
(559B-M0043-006) Heavy Chain Amino Acid Sequence (SEQ ID NO: 58)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSAYAMRWVRQAPGKGLEWVSYISSSGGETMYADSV
KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCANGYGRIDYWGQGTLVTVSS
>559B-R0048-A07 (559B-M0043-006) Light Chain Amino Acid Sequence
(SEQ ID NO: 59)
QSVLTQPASVSGSPGQSITISCTGTSSDIGGYNYVSWYQQHPGKAPKLMIYEVSNRPSGVSNRF
SGSKSGNTASLTISGLQAEDEADYYCSSYTSGSTRVFGIGTRVTVL >559B-R0048-G01
(559B-M0003-A08) Heavy Chain Amino Acid Sequence (SEQ ID NO: 60)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSAYVMRWVRQAPGKGLEWVSSIGSSGGPTYYADSV
KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRGGSGSSHAFDIWGQGTMVTVSS
>559B-R0048-G01 (559B-M0003-A08) Light Chain Amino Acid Sequence
(SEQ ID NO: 61)
QDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFS
GSGSGTDFTLTISSLQPEDSGTYYCQQYNSFPLTFGGGTKVEIK >559B-R0048-G09
(559B-M0054-B11) Heavy Chain Amino Acid Sequence (SEQ ID NO: 62)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSYYGMNWVRQAPGKGLEWVSVISPSGGLTVYADSV
KGRFTISRDNSKNTLYLQMNSLRAEDTAMYYCATGFAVQHGGGAFDIWGQGTMVTVSS
>559B-R0048-G09 (559B-M0054-B11) Light Chain Amino Acid Sequence
(SEQ ID NO: 63)
QDIQMTQSPATLSMSPGERATLSCRASQSVTTYLAWYQQKPGQAPRLLIYDASIRATGVPARFS
GSGSGTDFTLTISRLEPEDFAVYYCQQRTIWPLTFGGGTKVEIK >559B-R0048-E07
(559B-M0067-G11) Heavy Chain Amino Acid Sequence (SEQ ID NO: 64)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSPYEMVWVRQAPGKGLEWVSSIVPSGGWTVYADSV
KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASPSGRGLAFDIWGQGTMVTVSS
>559B-R0048-E07 (559B-M0067-G11) Light Chain Amino Acid Sequence
(SEQ ID NO: 65)
QDIQMTQSPGILSLSPGERATLSCRASQSISSSYLAWYQQKPGQAPRLLIYGASSRATGVPDRF
SGSGSGTEFTLTISSLQPEDFATYYCLQQKSYPYTFGQGTKVEIK >559B-R0048-007
(559B-M0065-B10) Heavy Chain Amino Acid Sequence (SEQ ID NO: 66)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSKYFMTWVRQAPGKGLEWVSWISSSGGYTNYADSV
KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGAYYYDAFDIWGQGTMVTVSS
>559B-R0048-007 (559B-M0065-B10) Light Chain Amino Acid Sequence
(SEQ ID NO: 67)
QDIQMTQSPSSLSASVGDRVTITCRASQSIAIFLNWYQQTPGKPPKLLIYGASTLQSGVPSRFS
GSGSGADFTLTISNLQLEDFTTYYCQQSYSTLYTFGQGTKLEIK >559B-R0049-0O3
(559B-M0037-E08) Heavy Chain Amino Acid Sequence (SEQ ID NO: 68)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYSMSWVRQAPGKGLEWVSVISSSGGMTYYADSV
KGRFTISRDNSKNTLYLQMNSLRAEDTAMYYCARDYYGNMDVWGKGTTVTVSS
>559B-R0049-0O3 (559B-M0037-E08) Light Chain Amino Acid Sequence
(SEQ ID NO: 69)
QDIQMTQSPSSLSTSVGDRVTITCRTSQDISGALAWYQQKPGKAPRLLIFGASSLESGVPSRFS
GSGSGTDFTLTISSLQPEDFATYYCQQFNKYPLTFGGGTKVEIK >559B-R0049-E01
(559B-M0035-A01) Heavy Chain Amino Acid Sequence (SEQ ID NO: 70)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSWYTMGWVRQAPGKGLEWVSYIYPSGGYTMYADSV
KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCANPYSSGGYWGQGTLVTVSS
>559B-R0049-E01 (559B-M0035-A01) Light Chain Amino Acid Sequence
(SEQ ID NO: 71)
QDIQMTQSPLSLPVTPGEPASISCRSSQSLLDSNGYNYLDWFLQKPGQSPQLLIYLGFNRASGV
PDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPYTFGQGTKLEIT
>559B-R0048-G03 (559B-M0003-E08) Heavy Chain Amino Acid Sequence
(SEQ ID NO: 72)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSAYLMTWVRQAPGKGLEWVSGISPSGGITKYADSV
KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDIPNWIYGMDVWGQGTTVTVSS
>559B-R0048-G03 (559B-M0003-E08) Light Chain Amino Acid Sequence
(SEQ ID NO: 73)
QSALTQPPSVSVSPGQTASITCSGDKLGNKYASWYQQKPGQSPVLVIYQDRRRPSGIPERFSGS
NSGNTATLTISGTQAMDEADYYCQAWDSGVVFGGGTKLTVL >559B-R0048-G07
(559B-M0052-E02) Heavy Chain Amino Acid Sequence (SEQ ID NO: 74)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYLMLWVRQAPGKGLEWVSGISPSGGGTAYADSV
KGRFTISRDNSKNTLYLQMNSLRAEDMAVYYCAKVAYSGSYYYYYYMDVWGKGTTVTVSS
>559B-R0048-G07 (559B-M0052-E02) Light Chain Amino Acid Sequence
(SEQ ID NO: 75)
QDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFS
GSGSGTDFTLTISSLQPEDFATYYCQQSYSTHSITFGQGTRLEIK >559B-M0064-H02
Heavy Chain Amino Acid Sequence (SEQ ID NO: 76)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSQYIMGWVRQAPGKGLEWVSSIGSSGVTVYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGGVTVLHAFDIWGQGTMVTVSSASTKGPSV
FPLAPSSKS >559B-M0064-H02 Light Chain Amino Acid Sequence (SEQ
ID NO: 77)
QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKVPKLIIYEGNKRPSGVPDRF
SGSKAGNTASLTVSGLQAEDEADYYCTAYGGHSRFYVFGTGTKVTVLGQPKANP
Also within the scope of this disclosure are functional equivalents
of any of the exemplary antibodies listed above. Such a functional
equivalent may bind to the same epitope of a cleaved HMWK and/or
intact HMWK, or the sample epitope of LMWK as one of the above
listed exemplary antibodies. In some embodiments, the functional
equivalent competes against one of the above-listed exemplary
antibodies for binding to a target antigen.
In some embodiments, the functional equivalent comprises a V.sub.H
chain that includes a V.sub.H CDR1, a V.sub.H CDR2, and/or a
V.sub.H CDR3 at least 75% (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%,
or 99%) identical to the corresponding V.sub.H CDRs of one of the
above-listed exemplary antibodies. Alternatively or in addition,
the functional equivalent comprises a V.sub.L CDR1, a V.sub.L CDR2,
and/or a V.sub.L CDR3 at least 75% (e.g., 80%, 85%, 90%, 95%, 96%,
97%, 98%, or 99%) identical to the exemplary antibody as listed
above. In some embodiments, the functional equivalent has the same
heavy chain and/or light chain complementarity determining regions
(CDRs) as one of the above-listed exemplary antibodies.
Alternatively or in addition, the functional equivalent comprises a
V.sub.H chain at least 75% (e.g., 80%, 85%, 90%, 95%, 96%, 97%,
98%, or 99%) identical to the V.sub.H chain of an exemplary
antibody and/or a V.sub.L chain at least 75% (e.g., 80%, 85%, 90%,
95%, 96%, 97%, 98%, or 99%) identical to the V.sub.L chain of the
exemplary antibody.
In some instances, the functional equivalent may contain one or
more (e.g., up to 5, up to 3, or up to 1) conservative mutations in
one or more of the heavy chain CDRs, or one or more of the light
chain CDRs in an exemplary antibody, e.g., at positions where the
residues are not likely to be involved in interacting with a target
antigen.
Without further elaboration, it is believed that one skilled in the
art can, based on the above description, utilize the present
present disclosure to its fullest extent. The following specific
embodiments are, therefore, to be construed as merely illustrative,
and not limitative of the remainder of the disclosure in any way
whatsoever. All publications cited herein are incorporated by
reference for the purposes or subject matter referenced herein.
EXAMPLES
Example 1: Development of Immunoassays for Specific Detection of
Cleaved HMWK
An ELISA-based immunoassay screen was initially developed to
identify Fab fragments in a phage display library that bound to
cleaved or intact HMWK. In general, the assay conditions relied on
biotinylated intact or cleaved HMWK immobilized on streptavidin
coated 384-well assay plates, blocking using a bovine serum albumin
(BSA) blocking buffer, and contacting the immobilized HMWK with Fab
displayed on phage from an overnight culture in E. coli (detected
with anti-M13-HRP antibody).
As shown in FIG. 12, panel A, the selection was directed towards
obtaining 2-chain HMWK specific antibodies by first preforming a
negative selection of the library with an input of approximately
1.times.10.sup.12 phage against biotinylated 1-chain HMWK
immobilized streptavidin coated magnetic beads (Dynabeads M280,
Thermo Fisher). The depleted library was then contacted with
biotinylated 2-chain HMWK immobilized on streptavidin coated
magnetic beads. The beads were extensively washed with PBS buffer
and used to infect E. coli for phage output amplification to
complete a round of selection. Three rounds of selection were
performed prior to screening individual phage colonies by ELISA
with biotinylated 1-chain and 2-chain HMWK immobilized on
streptavidin coated plates followed by detection with horse radish
peroxidase (HRP) conjugated anti-M13 antibody and absorbance
detection due to substrate hydrolysis for
3,3',5,5'-Tetramethylbenzidine (TMB). Recombinant Fab fragments
were expressed in E. coli and purified by protein A sepharose
chromatography (Wassaf et al. Anal. Biochem. (2006) 351: 241-253).
The specificity of each purified Fab was determined by coating 384
well plates and measuring binding to biotinylated 1-chain HMWK, to
biotinylated 2-chain HMWK, or to biotinylated LMWK, followed by
detection with streptavidin conjugated to HRP and TMB detection.
These assay conditions led to the identification of the
559B-M004-B04 isolate, which specifically binds cleaved HMWK over
intact HMWK (FIG. 1).
The immobilized HMWK was also contacted with a crude (unpurified)
559B-M004-B04 Fab preparation from an overnight culture in E. coli.
Fab bound to the HMWK was detected using an anti-human Fab-HRP
antibody, but did not result in specific binding to cleaved HMWK
(FIG. 1).
The configuration of the immunoassay was reversed by passively
immobilizing the purified Fab fragment of 559B-M004-B04 on
polystyrene 384-well assay plate. The Fab was contacted with
biotinylated HMWK, and the bound HMWK were detected with
streptavidin-HRP. (FIG. 1).
Unexpectedly, the specificity of the 559B-M004-B04 Fab to cleaved
HMWK was enhanced when the BSA blocking buffer was replaced with a
commercially available blocking buffer, the LowCross Blocking
Solution from Candor Biosciences during the initial screening
analyses (FIG. 1). Further, performing the immunoassay using
96-well assay plates rather than 384-well plates further increased
the observed specificity of 559B-M004-B04 to cleaved HMWK (FIG.
1).
The results obtained using the 559B-M004-B04 isolate led to the
development of an immunoassay (ELISA) for the detection of 2-HMWK
in samples (FIG. 12, panel B). This assay can also be used to
further evaluate binding characteristics of other Fab fragments and
antibodies. Briefly, a Fab is coated on to a multiwell plate
overnight. The following day the plate is washed then blocked with
BSA Buffer. Following a wash samples, standards, and QCs diluted in
LowCross Buffer are added to the plate and after a subsequent
incubation and then wash, any bound 2-Chain HMWK is detected by
adding HRP-labeled sheep anti-HMWK polyclonal detection antibody.
Following incubation with the detection antibody, the plate is
washed and TMB substrate is added to the plate. After a short
incubation the reaction is stopped with phosphoric acid. The
optical density is then measured at 450 nm-630 nm.
Example 2: Evaluation of Binding Specificity of Fab Clones Using
Immunoassays Described Herein
Thirty-six purified Fab clones (see Table 2 below) were assessed
for binding to cleaved HWMK, intact HMWK, and LWMK using the
immunoassay described in Example 1. Specifically, each of the
purified Fab clones was immobilized on 96-well assay plates at a
concentration of 1 .mu.g/L) in a total volume of 100 .mu.L in PBS
and incubated overnight at 2-8.degree. C. The assay plates were
blocked using LowCross blocking buffer. Biotinylated intact HMWK,
biotinylated cleaved HMWK or biotinylated LMWK (1 .mu.g/L each) was
added to each well in a total volume of 100 .mu.L and incubated for
2 hours prior to washing with a wash buffer. HRP-labeled
streptavidin was added to each well at a concentration of 100
ng/mL, and the signal was developed using Ultra TMB Substrate. The
signal to noise ratio was calculated using the signal observed upon
the addition of the biotinylated protein to an uncoated well. (FIG.
2, panels A and B). Based on the ELISA results, the antibodies can
be divided among 5 categories (Table 2).
TABLE-US-00007 TABLE 2 Binding characteristics of Fab fragments
ELISA Binding Fab Fragment Low affinity binder 559B-M0035-A01,
559B-M0052-E02, 559B-M0003-E08 Bind to cleaved and 559B-M0067-E02,
559B-M0039-G07, intact HMWK, not LMWK 559B-M0044-E09,
559B-M0003-C08, 559B-M0039-H06, 559B-M0039-D08, 559B-M0068-C07,
559B-M0021-G11, 559B-M0061-G06, 559B-M0036-G12, 559B-M0042-E06,
559B-M0070-H10, 559B-M0068-D01, 559B-M0004-E08 Bind to cleaved and
559B-M0069-C09, 559B-M0038-F04, intact HMWK and 559B-M0044-C05,
559B-M0047-H01, LMWK 559B-M0019-E12, 559B-X0004-B05,
559B-M0048-D12, 559B-M0053-G01, 559B-M0038-H03, 559B-M0017-H08,
559B-M0035-F05, 559B-M0035-H09, 559B-M0043-C06, 559B-M0003-A08,
559B-M0054-B11, 559B-M0067-G11, 559B-M0065-B10, 559B-M0064-H02
Mainly bind to LMWK 559B-M0037-E08 Specifically Bind to
559B-M0004-B04 cleaved HMWK
Several antibodies were obtained that bound to both 1-chain,
2-chain HMWK and LMWK, such as 559B-M0064-H02. These antibodies are
likely to bind an epitope in domains 1 through 4, which are shared
between HMWK and LMWK. M070-H10 is an example of an antibody
presumed to bind an epitope shared between 1-chain and 2-chain HMWK
but not on LMWK. LMWK is a kininogen splice variant leads to a
truncated protein composed of domains 1 through 4 and part of
domain 5 (Colman et al. Blood (1997) 90: 3819-3843). Consequently,
antibodies such as M070-H10 are likely to bind domain 5 or domain
6.
As shown in FIG. 14, panel A, 559B-M0004-B04 exhibited selectivity
for 2-chain over both 1-chain HMWK and LMWK and was selected for
further assay optimization. A sandwich ELISA was developed to
detect cleaved HMWK in human plasma samples in which 559B-M0004-B04
(100 .mu.L of 2 .mu.g/mL) was passively immobilized on a 96 well
plate (Nunc Maxisorp plate) (FIG. 12, panel B). The following day,
the plate was washed and then blocked with 2% BSA (Protease/IgG
free) in PBS buffer. Following a wash, samples containing cleaved
HMWK in 0.1% BSA buffer in PBS with 0.05% Tween-20 (2-Chain HMWK
assay buffer). Purified protein standards (e.g., 2-chain HMWK,
intact HMWK or LMWK) were spiked into HNKW HMWK-deficient plasma
and diluted 1:320 in 2-chain HMWK assay buffer. Following plate
washing with PBST, a mixture of 2 mouse monoclonal antibodies
(11H05 and 13B12) at 1 .mu.g/mL in 2-chain HMWK assay buffer were
added for 1 hour at room temperature. Unbound detection antibodies
were washed and a 1:2000 dilution of goat anti-mouse secondary
antibody conjugated to horseradish peroxidase (HRP) was added. The
assay containing the secondary antibody was incubated for 1 hour at
room temperature, and unbound secondary antibody was removed by
washing with 2-chain HMWK assay buffer. Signal was detected by the
addition of 3, 3',5,5'-tetramethylbenzidine (TMB), an HRP
substrate. The reaction was stopped with phosphoric acid.
Hydrolysis of a TMB substrate was detected using a microplate
reader at 450 nm-630 nm (FIG. 3). Additionally, performing the
ELISA assay using samples containing cleaved HMWK in 2-chain HMWK
assay buffer buffer or HMWK-deficient plasma and analyzed in the
presence of either 2.5% or 10% plasma resulted in similar binding
(FIG. 4). Using these immunoassay conditions, specifically binding
to cleaved HMWK was detected. The assay resulted in comparable
performance when HMWK was provided in either 2-chaim HMWK assay
buffer or HMWK-deficient plasma (FIGS. 3 and 4). Furthermore, there
was no binding of 559B-M0004-B04 to LMWK.
The ELISA assay was evaluated for detection of cleaved HMWK
generated upon contact activation in human plasma (FIGS. 5A and
5B). The amount of cleaved HMWK in normal human plasma was measured
in the absence or presence of a catalytic amount of FXIIa, pKal, or
ellagic acid, which causes FXII auto-activation to FXIIa and
consequently generation of cleaved HMWK (FIG. 5, panels A and B).
Consistent with the role of plasma kallikrein as the primary plasma
enzyme required for the generation of 2-chain HMWK, neither ellagic
acid nor FXIIa addition lead to the generation of cleaved HMWK in
prekallikrein-deficient plasma. The contact system in FXI deficient
plasma was equally activated using either FXIIa, pKal, or ellagic;
a result consistent with the understanding that FXIa is generated
by FXIIa and does not produce 2-chain HMWK.
The results from the 2-Chain HMWK ELISA were corroborated by
detecting cleaved HMWK generated upon contact activation in human
plasma by Western blot analysis using the mouse monoclonal
antibody, 11H05 (FIG. 10). The 11H05 antibody specifically binds
the light chain of HMWK and illuminates both the 56 kDa light chain
and the further proteolyzed 46 kDa light chain, which is
subsequently generated through the proteolytic activity of plasma
kallikrein at a site near the N-terminus of the HMWK light chain
(Colman et al. Blood (1997) 90: 3819-3843).
The ELISA assay was also evaluated for the ability to detect
cleaved HMWK generated in plasma from 12 normal donors (FIG. 6).
Following ellagic acid activation of the contact activation system,
cleaved HMWK was detected in each of the 12 samples. The amount of
cleaved HMWK was also measured after the contact activation system
was inhibited in normal plasma using various concentrations of
landadelumab (DX-2930; a specific inhibitor of plasma kallikrein)
or an inhibitor of the serpin C1-INH, then activated with ellagic
acid (FIG. 7, panels A and B). Landadelumab (DX-2930) is a fully
human antibody potent (K.sub.1=0.12 nM) and specific inhibitor of
plasma kallikrein that was discovered using phage and is in
clinical development for the prophylactic treatment of HAE-C1INH
attacks (Chyung et al. Ann. Allergy Asthma Immunol. (2014) 113:
460-466; Kenniston et al. J. Biol. Chem. (1994) 289: 23596-23608).
When lanadelumab was spiked into citrated plasma at different
concentrations it effectively inhibited the generation of 2-chain
HMWK induced by FXIIa as shown by Western blot and sandwich ELISA
(FIG. 7B). The IC.sub.50 for lanadelumab inhibition of 2-chain HMWK
generation was 212.+-.28 nM, which is consistent with the value
expected for the activation of all prekallikrein in neat plasma
(approximately 500 nM). The complete inhibition of signal by
landadelumab in plasma treated with a contact system activator
confirms that M004-B04 is specific for 2-chain HMWK generated by
plasma kallikrein.
Activation of the contact system in kininogen-deficient plasma did
not yield an increase in ELISA signal in this preliminary assay
using M004-B04 as the capture antibody and a HRP-conjugated sheep
polyclonal anti-kininogen as the detection antibody (data not
shown).
It is also evident from FIG. 10 that plasma collected from a
healthy subject using EDTA as an anti-coagulant was activated
similarly as citrated plasma; supporting the observation that metal
ions are not required for contact system activation (Colman et al.
Blood (1997) 90: 3819-3843). However, 2-chain HMWK was not detected
by ELISA in EDTA plasma (FIG. 5B) suggesting that M004-B04 antibody
binding to 2-chain HMWK is dependent upon a metal ion. A zinc
binding site on HMWK in domain 5 (amino acids 479-498) of the light
chain was previously identified and shown to mediate kininogen
interactions with the endothelial cell surface receptors gC1qR,
cytokeratin 1, and the urokinase plasminogen activator receptor and
thereby enhance contact system activation (Kaplan et al. Adv.
Immunol. (2014) 121: 41-89; Bjorkqvist et al. Biol. Chem. (2013)
394: 1195-1204). The addition of ZnCl.sub.2 to the assay buffer was
tested at various concentrations and was found to enhance binding
of the antibody to cleaved HMWK (FIG. 11). Increasing
concentrations of ZnCl2 on the ELISA signal observed with ellagic
acid activated citrated and EDTA plasma was investigated. The ELISA
signal in EDTA plasma increased to an apparent maximum at
ZnCl.sub.2 concentrations above 400 .mu.M (in well
concentration).
Binding of 1-chain HMWK to zinc was previously shown using electron
microscopy to promote a more compact and spherical quaternary
structure (Herwald et al. Eur J. Biochem. (2001) 268: 396-404). It
was also shown by electron microscopy that 2-chain HMWK adopts a
more elongated, less spherical, quaternary structure than 1-chain
HMWK in a buffer containing EDTA (Herwald et al. Eur J. Biochem.
(2001) 268: 396-404). Though the effect of zinc on the structure of
2-chain HMWK was not previously reported, the apparent zinc
dependent binding of M004-B04 described herein suggests the 2-chain
HMWK exists in a unique conformation in the presence of zinc.
The EDTA concentration in plasma collected in commercially
available spray coated K.sub.2EDTA tubes is approximately 4 mM,
which following a 1:20 dilution converts to an in-well
concentration of approximately 200 .mu.M and is consistent with the
restoration of Zinc-dependent binding upon addition of sufficient
ZnCl.sub.2 to overwhelm the chelating capacity of EDTA. In
contrast, the ELISA signal citrated plasma activated using ellagic
acid was not increased in the presence of 25 or 50 .mu.M ZnCl.sub.2
(in well concentrations) but at concentrations above 100 .mu.M
ZnCl.sub.2 the ELISA signal increased to a maximum above 200 .mu.M
ZnCl.sub.2. (FIG. 11) The normal concentration for zinc in plasma
from healthy volunteers is 10-17 .mu.M (Wessells et al. J. Nutr.
(2014) 144: 1204-1210). Since the ELISA signal observed in
activated citrated plasma only increased when with in-well
ZnCl.sub.2 concentrations >50 .mu.M, which would equates to
in-plasma concentrations >1 mM, it appears that the ELISA is not
susceptible to physiologic fluctuations in the concentration of
zinc in the plasma. Consequently, the subsequent experiments did
not add ZnCl.sub.2 to the assay buffer.
As described above, the binding of 559B-M004-B04 to 2-chain HMWK
was enhanced by supra-physiologic concentrations of ZnCl.sub.2 and
inhibited by metal chelation with high concentrations of EDTA. A
zinc binding site has been described in domain of 2-chain HWMK and
a synthetic peptide encompassing this site (HKH20,
HKHGHGHGKHKNKGKKNGKH (SEQ ID NO: 83) was shown to inhibit contact
system activation via an attenuation of cell surface association
(Nakazawa et al. Int. Immunopharmacol. (2002) 2: 1875-1885).
Consequently, the HKH20 peptide, as well as the GCP28 peptide
corresponding to sequences in domain 3 were tested for their
ability to inhibit 2-chain HMWK binding to 559B-M004-B04 by ELISA.
As shown in FIG. 15, the HKH20 peptide but not the GCP28 peptide
inhibits 2-chain HMWK binding to M004-B04, which suggests that the
M004-B04 epitope could reside within domain 5 in the vicinity of
the zinc binding site. To perform the assay, the kininogen peptides
were diluted to 250 .mu.g/mL and allowed to preincubate on assay
plate. Then, purified 2-chain HMWK in deficient human plasma was
diluted 160 and then added to plate.
Time dependence of generation of cleaved HMWK in normal citrated
human plasma was assessed at various time points following
activation of the contact activation system with ellagic acid or
FXIIa (FIG. 8). Finally, the ELISA assay was used to assess the
presence and quantity of cleaved HMWK in plasma samples from
patients with hereditary angioedema (HAE) compared to citrated
plasma samples from normal patients (without HAE). The samples from
patients with HAE were found to contain elevated levels of 2-chain
HMWK (1423.+-.603 ng/mL) relative to samples from normal donors
(432.4.+-.186 ng/mL) (FIG. 9), which are statistically different
(P=0.017) by one way ANOVA analysis.
Having determined that M004-B04 specifically binds a neo-epitope on
2-chain HMWK that is not present on 1-chain HMWK or LMWK and
demonstrating that the antibody binding is dependent on plasma
kallikrein activity, the assay was also tested using a pair of
mouse monoclonal antibodies (11H05 and 13B12) for the detection
(FIG. 16). Antibody 13B12 appears to bind the heavy chain of HMWK
and 11H05 appears to bind the light chain of HMWK, the combination
of both antibodies for detection resulted in a signal boost,
possibly due to their non-overlapping binding epitopes in the
antigen.
The importance of plasma collection on the assessment of contact
system has been previously described (Suffritti et al. Clin. Exp.
Allergy (2014) 44: 1503-1514). It is well known that contact of
plasma with glass or other polar surfaces results in extensive ex
vivo contact system activation that can mask the accurate
determination of endogenous contact system activation (Colman et
al. Blood (1997) 90: 3819-3843). The ability of the optimized
sandwich ELISA to detect 2-chain HMWK was compared in different
plasma types, including a customized plasma containing a mixture of
protease inhibitors in acid citrate dextrose in an evacuated,
plastic blood collection tube referred to as SCAT169 (HTI, Essex
Vt). As shown in FIG. 6, the standard curve prepared in SCAT169
plasma is less sensitive than the curve prepared in citrated
plasma, likely due to the inclusion of 2 mM EDTA in the collected
plasma. At the plasma dilution used in this assay (1:320) this
concentration of EDTA (3.1 .mu.M) does not interfere significantly
with the 2-chain HMWK and may assist in stabilizing the plasma from
proteolytic degradation due to metalloproteases.
Citrated and SCAT169 plasma from healthy volunteers was compared to
samples from HAE patients by Western blot and the sandwich ELISA
assay. In FIG. 17, panels A-C, the Western blot method of detecting
2-chain HMWK (i.e. cleaved kininogen) in citrated plasma was
capable of differentiating samples from HAE patients from healthy
volunteers (HV), as shown by receiver operator characteristic (ROC)
analysis with an area under the curve (AUC) value of 0.977 for the
comparison of basal to HV, or 1.0 for the comparison of attack to
HV. Citrated plasma samples from HAE patients during quiescence
(basal) were differentiated from attack samples with an AUC of
0.625 (FIG. 17, panel D).
As shown in FIG. 18, panels A-C, the Western blot method of
detecting 2-chain HMWK in SCAT169 plasma was capable of
differentiating samples from HAE patients from samples from healthy
volunteers (HV), as shown by ROC analysis an AUC value of 0.915 for
the comparison of basal to HV, or 0.967 for the comparison of
attack to HV. SCAT169 samples from HAE patients during quiescence
(basal) were differentiated from from attack samples with an AUC of
0.597 (FIG. 18, panel D).
In FIG. 19, panels A-C, the 2-chain ELISA method of detecting
2-chain HMWK in citrated plasma was capable of differentiating
samples from HAE patients from healthy volunteers, as shown by ROC
analysis with an AUC value of 0.915 for the comparison of basal to
HV, or 0.866 for the comparison of attack to HV. Citrated plasma
samples from HAE patients during quiescence (basal) were
differentiated from from attack samples with an AUC of 0.709 (FIG.
19, panel D).
As shown in FIG. 20, panels A-C. the 2-chain ELISA method of
detecting 2-chain HMWK in SCAT169 samples was capable of
differentiating samples from HAE patients from healthy volunteers,
as shown by ROC analysis with an AUC value of 0.999 for the
comparison of basal to HV, or 1.0 for the comparison of attack to
HV. Citrated plasma samples from HAE patients during quiescence
(basal) were differentiated from from attack samples with an AUC of
0.8176 (FIG. 20, panel D).
For the above ROC analysis, both the 2-chain HMWK Western blot and
the 2-chain HMWK ELISA demonstrated herein may be useful in
differentiating patients having or at risk of having HAE based on
the levels of cleaved kininogen in plasma, as compared to healthy
volunteers. The presence of protease inhibitors in SCAT169 plasma
reduced the ex vivo plasma activation during blood collection.
OTHER EMBODIMENTS
All of the features disclosed in this specification may be combined
in any combination. Each feature disclosed in this specification
may be replaced by an alternative feature serving the same,
equivalent, or similar purpose. Thus, unless expressly stated
otherwise, each feature disclosed is only an example of a generic
series of equivalent or similar features.
From the above description, one skilled in the art can easily
ascertain the essential characteristics of the present disclosure,
and without departing from the spirit and scope thereof, can make
various changes and modifications of the present disclosure to
adapt it to various usages and conditions. Thus, other embodiments
are also within the claims.
EQUIVALENTS AND SCOPE
Those skilled in the art will recognize, or be able to ascertain
using no more than routine experimentation, many equivalents to the
specific embodiments of the present disclosure described herein.
The scope of the present disclosure is not intended to be limited
to the above description, but rather is as set forth in the
appended claims.
In the claims articles such as "a," "an," and "the" may mean one or
more than one unless indicated to the contrary or otherwise evident
from the context. Claims or descriptions that include "or" between
one or more members of a group are considered satisfied if one,
more than one, or all of the group members are present in, employed
in, or otherwise relevant to a given product or process unless
indicated to the contrary or otherwise evident from the context.
The present disclosure includes embodiments in which exactly one
member of the group is present in, employed in, or otherwise
relevant to a given product or process. The present disclosure
includes embodiments in which more than one, or all of the group
members are present in, employed in, or otherwise relevant to a
given product or process.
Furthermore, the present disclosure encompasses all variations,
combinations, and permutations in which one or more limitations,
elements, clauses, and descriptive terms from one or more of the
listed claims is introduced into another claim. For example, any
claim that is dependent on another claim can be modified to include
one or more limitations found in any other claim that is dependent
on the same base claim. Where elements are presented as lists,
e.g., in Markush group format, each subgroup of the elements is
also disclosed, and any element(s) can be removed from the group.
It should it be understood that, in general, where the present
disclosure, or aspects of the present disclosure, is/are referred
to as comprising particular elements and/or features, certain
embodiments of the present disclosure or aspects of the present
disclosure consist, or consist essentially of, such elements and/or
features. For purposes of simplicity, those embodiments have not
been specifically set forth in haec verba herein. It is also noted
that the terms "comprising" and "containing" are intended to be
open and permits the inclusion of additional elements or steps.
Where ranges are given, endpoints are included. Furthermore, unless
otherwise indicated or otherwise evident from the context and
understanding of one of ordinary skill in the art, values that are
expressed as ranges can assume any specific value or sub-range
within the stated ranges in different embodiments of the present
disclosure, to the tenth of the unit of the lower limit of the
range, unless the context clearly dictates otherwise.
This application refers to various issued patents, published patent
applications, journal articles, and other publications, all of
which are incorporated herein by reference. If there is a conflict
between any of the incorporated references and the instant
specification, the specification shall control. In addition, any
particular embodiment of the present present disclosure that falls
within the prior art may be explicitly excluded from any one or
more of the claims. Because such embodiments are deemed to be known
to one of ordinary skill in the art, they may be excluded even if
the exclusion is not set forth explicitly herein. Any particular
embodiment of the present disclosure can be excluded from any
claim, for any reason, whether or not related to the existence of
prior art.
Those skilled in the art will recognize or be able to ascertain
using no more than routine experimentation many equivalents to the
specific embodiments described herein. The scope of the present
embodiments described herein is not intended to be limited to the
above Description, but rather is as set forth in the appended
claims. Those of ordinary skill in the art will appreciate that
various changes and modifications to this description may be made
without departing from the spirit or scope of the present
disclosure, as defined in the following claims.
SEQUENCE LISTINGS
1
831644PRTHomo sapiens 1Met Lys Leu Ile Thr Ile Leu Phe Leu Cys Ser
Arg Leu Leu Leu Ser1 5 10 15Leu Thr Gln Glu Ser Gln Ser Glu Glu Ile
Asp Cys Asn Asp Lys Asp 20 25 30Leu Phe Lys Ala Val Asp Ala Ala Leu
Lys Lys Tyr Asn Ser Gln Asn 35 40 45Gln Ser Asn Asn Gln Phe Val Leu
Tyr Arg Ile Thr Glu Ala Thr Lys 50 55 60Thr Val Gly Ser Asp Thr Phe
Tyr Ser Phe Lys Tyr Glu Ile Lys Glu65 70 75 80Gly Asp Cys Pro Val
Gln Ser Gly Lys Thr Trp Gln Asp Cys Glu Tyr 85 90 95Lys Asp Ala Ala
Lys Ala Ala Thr Gly Glu Cys Thr Ala Thr Val Gly 100 105 110Lys Arg
Ser Ser Thr Lys Phe Ser Val Ala Thr Gln Thr Cys Gln Ile 115 120
125Thr Pro Ala Glu Gly Pro Val Val Thr Ala Gln Tyr Asp Cys Leu Gly
130 135 140Cys Val His Pro Ile Ser Thr Gln Ser Pro Asp Leu Glu Pro
Ile Leu145 150 155 160Arg His Gly Ile Gln Tyr Phe Asn Asn Asn Thr
Gln His Ser Ser Leu 165 170 175Phe Met Leu Asn Glu Val Lys Arg Ala
Gln Arg Gln Val Val Ala Gly 180 185 190Leu Asn Phe Arg Ile Thr Tyr
Ser Ile Val Gln Thr Asn Cys Ser Lys 195 200 205Glu Asn Phe Leu Phe
Leu Thr Pro Asp Cys Lys Ser Leu Trp Asn Gly 210 215 220Asp Thr Gly
Glu Cys Thr Asp Asn Ala Tyr Ile Asp Ile Gln Leu Arg225 230 235
240Ile Ala Ser Phe Ser Gln Asn Cys Asp Ile Tyr Pro Gly Lys Asp Phe
245 250 255Val Gln Pro Pro Thr Lys Ile Cys Val Gly Cys Pro Arg Asp
Ile Pro 260 265 270Thr Asn Ser Pro Glu Leu Glu Glu Thr Leu Thr His
Thr Ile Thr Lys 275 280 285Leu Asn Ala Glu Asn Asn Ala Thr Phe Tyr
Phe Lys Ile Asp Asn Val 290 295 300Lys Lys Ala Arg Val Gln Val Val
Ala Gly Lys Lys Tyr Phe Ile Asp305 310 315 320Phe Val Ala Arg Glu
Thr Thr Cys Ser Lys Glu Ser Asn Glu Glu Leu 325 330 335Thr Glu Ser
Cys Glu Thr Lys Lys Leu Gly Gln Ser Leu Asp Cys Asn 340 345 350Ala
Glu Val Tyr Val Val Pro Trp Glu Lys Lys Ile Tyr Pro Thr Val 355 360
365Asn Cys Gln Pro Leu Gly Met Ile Ser Leu Met Lys Arg Pro Pro Gly
370 375 380Phe Ser Pro Phe Arg Ser Ser Arg Ile Gly Glu Ile Lys Glu
Glu Thr385 390 395 400Thr Val Ser Pro Pro His Thr Ser Met Ala Pro
Ala Gln Asp Glu Glu 405 410 415Arg Asp Ser Gly Lys Glu Gln Gly His
Thr Arg Arg His Asp Trp Gly 420 425 430His Glu Lys Gln Arg Lys His
Asn Leu Gly His Gly His Lys His Glu 435 440 445Arg Asp Gln Gly His
Gly His Gln Arg Gly His Gly Leu Gly His Gly 450 455 460His Glu Gln
Gln His Gly Leu Gly His Gly His Lys Phe Lys Leu Asp465 470 475
480Asp Asp Leu Glu His Gln Gly Gly His Val Leu Asp His Gly His Lys
485 490 495His Lys His Gly His Gly His Gly Lys His Lys Asn Lys Gly
Lys Lys 500 505 510Asn Gly Lys His Asn Gly Trp Lys Thr Glu His Leu
Ala Ser Ser Ser 515 520 525Glu Asp Ser Thr Thr Pro Ser Ala Gln Thr
Gln Glu Lys Thr Glu Gly 530 535 540Pro Thr Pro Ile Pro Ser Leu Ala
Lys Pro Gly Val Thr Val Thr Phe545 550 555 560Ser Asp Phe Gln Asp
Ser Asp Leu Ile Ala Thr Met Met Pro Pro Ile 565 570 575Ser Pro Ala
Pro Ile Gln Ser Asp Asp Asp Trp Ile Pro Asp Ile Gln 580 585 590Ile
Asp Pro Asn Gly Leu Ser Phe Asn Pro Ile Ser Asp Phe Pro Asp 595 600
605Thr Thr Ser Pro Lys Cys Pro Gly Arg Pro Trp Lys Ser Val Ser Glu
610 615 620Ile Asn Pro Thr Thr Gln Met Lys Glu Ser Tyr Tyr Phe Asp
Leu Thr625 630 635 640Asp Gly Leu Ser2362PRTHomo sapiens 2Gln Glu
Ser Gln Ser Glu Glu Ile Asp Cys Asn Asp Lys Asp Leu Phe1 5 10 15Lys
Ala Val Asp Ala Ala Leu Lys Lys Tyr Asn Ser Gln Asn Gln Ser 20 25
30Asn Asn Gln Phe Val Leu Tyr Arg Ile Thr Glu Ala Thr Lys Thr Val
35 40 45Gly Ser Asp Thr Phe Tyr Ser Phe Lys Tyr Glu Ile Lys Glu Gly
Asp 50 55 60Cys Pro Val Gln Ser Gly Lys Thr Trp Gln Asp Cys Glu Tyr
Lys Asp65 70 75 80Ala Ala Lys Ala Ala Thr Gly Glu Cys Thr Ala Thr
Val Gly Lys Arg 85 90 95Ser Ser Thr Lys Phe Ser Val Ala Thr Gln Thr
Cys Gln Ile Thr Pro 100 105 110Ala Glu Gly Pro Val Val Thr Ala Gln
Tyr Asp Cys Leu Gly Cys Val 115 120 125His Pro Ile Ser Thr Gln Ser
Pro Asp Leu Glu Pro Ile Leu Arg His 130 135 140Gly Ile Gln Tyr Phe
Asn Asn Asn Thr Gln His Ser Ser Leu Phe Met145 150 155 160Leu Asn
Glu Val Lys Arg Ala Gln Arg Gln Val Val Ala Gly Leu Asn 165 170
175Phe Arg Ile Thr Tyr Ser Ile Val Gln Thr Asn Cys Ser Lys Glu Asn
180 185 190Phe Leu Phe Leu Thr Pro Asp Cys Lys Ser Leu Trp Asn Gly
Asp Thr 195 200 205Gly Glu Cys Thr Asp Asn Ala Tyr Ile Asp Ile Gln
Leu Arg Ile Ala 210 215 220Ser Phe Ser Gln Asn Cys Asp Ile Tyr Pro
Gly Lys Asp Phe Val Gln225 230 235 240Pro Pro Thr Lys Ile Cys Val
Gly Cys Pro Arg Asp Ile Pro Thr Asn 245 250 255Ser Pro Glu Leu Glu
Glu Thr Leu Thr His Thr Ile Thr Lys Leu Asn 260 265 270Ala Glu Asn
Asn Ala Thr Phe Tyr Phe Lys Ile Asp Asn Val Lys Lys 275 280 285Ala
Arg Val Gln Val Val Ala Gly Lys Lys Tyr Phe Ile Asp Phe Val 290 295
300Ala Arg Glu Thr Thr Cys Ser Lys Glu Ser Asn Glu Glu Leu Thr
Glu305 310 315 320Ser Cys Glu Thr Lys Lys Leu Gly Gln Ser Leu Asp
Cys Asn Ala Glu 325 330 335Val Tyr Val Val Pro Trp Glu Lys Lys Ile
Tyr Pro Thr Val Asn Cys 340 345 350Gln Pro Leu Gly Met Ile Ser Leu
Met Lys 355 3603255PRTHomo sapiens 3Ser Ser Arg Ile Gly Glu Ile Lys
Glu Glu Thr Thr Val Ser Pro Pro1 5 10 15His Thr Ser Met Ala Pro Ala
Gln Asp Glu Glu Arg Asp Ser Gly Lys 20 25 30Glu Gln Gly His Thr Arg
Arg His Asp Trp Gly His Glu Lys Gln Arg 35 40 45Lys His Asn Leu Gly
His Gly His Lys His Glu Arg Asp Gln Gly His 50 55 60Gly His Gln Arg
Gly His Gly Leu Gly His Gly His Glu Gln Gln His65 70 75 80Gly Leu
Gly His Gly His Lys Phe Lys Leu Asp Asp Asp Leu Glu His 85 90 95Gln
Gly Gly His Val Leu Asp His Gly His Lys His Lys His Gly His 100 105
110Gly His Gly Lys His Lys Asn Lys Gly Lys Lys Asn Gly Lys His Asn
115 120 125Gly Trp Lys Thr Glu His Leu Ala Ser Ser Ser Glu Asp Ser
Thr Thr 130 135 140Pro Ser Ala Gln Thr Gln Glu Lys Thr Glu Gly Pro
Thr Pro Ile Pro145 150 155 160Ser Leu Ala Lys Pro Gly Val Thr Val
Thr Phe Ser Asp Phe Gln Asp 165 170 175Ser Asp Leu Ile Ala Thr Met
Met Pro Pro Ile Ser Pro Ala Pro Ile 180 185 190Gln Ser Asp Asp Asp
Trp Ile Pro Asp Ile Gln Ile Asp Pro Asn Gly 195 200 205Leu Ser Phe
Asn Pro Ile Ser Asp Phe Pro Asp Thr Thr Ser Pro Lys 210 215 220Cys
Pro Gly Arg Pro Trp Lys Ser Val Ser Glu Ile Asn Pro Thr Thr225 230
235 240Gln Met Lys Glu Ser Tyr Tyr Phe Asp Leu Thr Asp Gly Leu Ser
245 250 2554123PRTArtificial SequenceSynthetic Polypeptide 4Glu Val
Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Phe Tyr 20 25
30Val Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ser Gly Ile Ser Pro Ser Gly Gly Asn Thr Ala Tyr Ala Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Arg Lys Leu Phe Tyr Tyr Asp Asp Thr Lys
Gly Tyr Phe Asp Phe 100 105 110Trp Gly Gln Gly Thr Leu Val Thr Val
Ser Ser 115 1205110PRTArtificial SequenceSynthetic Polypeptide 5Gln
Tyr Glu Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln1 5 10
15Arg Val Thr Leu Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn
20 25 30Tyr Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu
Leu 35 40 45Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg
Phe Ser 50 55 60Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser
Gly Leu Gln65 70 75 80Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala
Trp Asp Asp Ser Leu 85 90 95Asn Gly Arg Val Phe Gly Gly Gly Thr Lys
Leu Thr Val Leu 100 105 1106120PRTArtificial SequenceSynthetic
Polypeptide 6Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Leu Tyr 20 25 30Pro Met Val Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu Glu Trp Val 35 40 45Ser Ser Ile Tyr Pro Ser Gly Gly Phe Thr
Thr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Ser Ser Arg Tyr
Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln 100 105 110Gly Thr Thr Val
Thr Val Ser Ser 115 1207110PRTArtificial SequenceSynthetic
Polypeptide 7Gln Tyr Glu Leu Thr Gln Pro Pro Ser Met Ser Gly Thr
Pro Gly Gln1 5 10 15Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn
Ile Gly Ser Glu 20 25 30Tyr Val Tyr Trp Phe Gln Gln Leu Pro Gly Thr
Ala Pro Lys Leu Leu 35 40 45Ile Tyr Arg Asn Asp Gln Arg Pro Ser Gly
Val Pro Asp Arg Phe Ser 50 55 60Gly Ser Lys Ser Gly Thr Ser Ala Ser
Leu Ala Ile Ser Gly Leu Arg65 70 75 80Ser Glu Asp Glu Thr Asp Tyr
Tyr Cys Ser Thr Trp Asp Asp Thr Leu 85 90 95Arg Thr Gly Val Phe Gly
Gly Gly Thr Lys Val Thr Val Leu 100 105 1108118PRTArtificial
SequenceSynthetic Polypeptide 8Glu Val Gln Leu Leu Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Arg Tyr 20 25 30Arg Met Arg Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Gly Ile Ser Pro Ser
Gly Gly Trp Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Thr Thr
Asp Asn Gly Asp Tyr Ala Leu Ala His Trp Gly Gln Gly Thr 100 105
110Leu Val Thr Val Ser Ser 1159108PRTArtificial SequenceSynthetic
Polypeptide 9Gln Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser
Ala Ser Val1 5 10 15Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
Arg Ile Ile Asn 20 25 30Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys
Ala Pro Lys Leu Leu 35 40 45Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly
Val Pro Ser Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Ser Ser Leu Gln65 70 75 80Pro Glu Asp Phe Ala Thr Tyr
Tyr Cys Gln Gln Ser Tyr Ser Ala Pro 85 90 95Leu Thr Phe Gly Gly Gly
Thr Arg Val Glu Ile Lys 100 10510125PRTArtificial SequenceSynthetic
Polypeptide 10Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Gln Tyr 20 25 30Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu Glu Trp Val 35 40 45Ser Ser Ile Tyr Ser Ser Gly Gly Ser Thr
Gln Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95Ala Arg Thr Arg Arg Gly
Trp Phe Gly Glu Asp Tyr Tyr Tyr Tyr Met 100 105 110Asp Val Trp Gly
Lys Gly Thr Thr Val Thr Val Ser Ser 115 120 12511108PRTArtificial
SequenceSynthetic Polypeptide 11Gln Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val1 5 10 15Gly Asp Arg Ile Thr Ile Thr Cys
Arg Ala Ser Gln Gly Ile Arg Asn 20 25 30Asp Val Gly Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Gln Arg Leu 35 40 45Ile Tyr Ala Ala Ser Ser
Leu Gln Ser Gly Val Pro Ser Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly
Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln65 70 75 80Pro Glu Asp
Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn Ser Tyr Pro 85 90 95Leu Thr
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 10512126PRTArtificial
SequenceSynthetic Polypeptide 12Glu Val Gln Leu Leu Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30Met Met Tyr Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ser Ile Ser Pro Ser
Gly Gly Lys Thr Trp Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg
Leu Gly Gly Ser Ser Ser Tyr Tyr Tyr Tyr Tyr Tyr Tyr Gly 100 105
110Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120
12513111PRTArtificial SequenceSynthetic Polypeptide 13Gln Ser Ala
Leu Thr Gln Ser Pro Ser Ala Ser Gly Thr Pro Gly Gln1 5 10 15Arg Val
Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Gly Asn 20 25 30Thr
Val Asn Trp Tyr Gln Gln Phe Pro Gly Thr Ala Pro Lys Leu Leu 35 40
45Ile Tyr Ser Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu
Gln65 70 75 80Ser Glu Asp Glu Ala Ile Tyr Tyr Cys Ala Ser Trp Asp
Asp Arg Leu 85 90 95Asn Gly
His Trp Val Phe Gly Gly Gly Thr Arg Leu Thr Val Leu 100 105
11014123PRTArtificial SequenceSynthetic Polypeptide 14Glu Val Gln
Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ala Tyr 20 25 30Asp
Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ser Ser Ile Trp Pro Ser Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val
50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95Ala Arg Gly Asp Tyr Asp Tyr Gly Asp Phe Thr Asp
Ala Phe Asp Ile 100 105 110Trp Gly Gln Gly Thr Met Val Thr Val Ser
Ser 115 12015110PRTArtificial SequenceSynthetic Polypeptide 15Gln
Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln1 5 10
15Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Ser Tyr
20 25 30Asn Leu Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys
Leu 35 40 45Met Ile Tyr Glu Gly Ser Lys Arg Pro Ser Gly Val Pro Asp
Arg Phe 50 55 60Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Ile Ile
Ser Gly Leu65 70 75 80Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Cys
Ser Tyr Ala Gly Ser 85 90 95Tyr Ser Tyr Val Phe Gly Thr Gly Thr Arg
Val Thr Val Leu 100 105 11016118PRTArtificial SequenceSynthetic
Polypeptide 16Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Asn Tyr 20 25 30Ala Met Gln Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu Glu Trp Val 35 40 45Ser Trp Ile Tyr Ser Ser Gly Gly Pro Thr
Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Leu Pro Gly
Gln Pro Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110Leu Val Thr Val
Ser Ser 11517110PRTArtificial SequenceSynthetic Polypeptide 17Gln
Ser Glu Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln1 5 10
15Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn
20 25 30Tyr Val Tyr Trp Tyr Gln Gln Phe Pro Gly Thr Ala Pro Lys Leu
Leu 35 40 45Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg
Phe Ser 50 55 60Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser
Gly Leu Arg65 70 75 80Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Thr
Trp Asp Asp Arg Leu 85 90 95Ser Gly Trp Val Phe Gly Gly Gly Thr Lys
Leu Thr Val Leu 100 105 11018116PRTArtificial SequenceSynthetic
Polypeptide 18Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Ser Tyr 20 25 30Gln Met His Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu Glu Trp Val 35 40 45Ser Gly Ile Tyr Ser Ser Gly Gly Ser Thr
Pro Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly His His Gly
Met Asp Val Trp Gly Gln Gly Thr Thr Val 100 105 110Thr Val Ser Ser
11519108PRTArtificial SequenceSynthetic Polypeptide 19Gln Asp Ile
Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val1 5 10 15Gly Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser 20 25 30Trp
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 35 40
45Ile Tyr Ala Ala Ser Asn Leu Gln Ser Gly Val Pro Ser Arg Phe Ser
50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Gln65 70 75 80Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Lys Tyr Asn
Ile Ala Pro 85 90 95Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 10520120PRTArtificial SequenceSynthetic Polypeptide 20Glu Val
Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25
30Pro Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ser Gly Ile Ser Ser Ser Gly Gly Phe Thr Pro Tyr Ala Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Arg Met Val Arg Gly Val Ile Lys Ala Phe
Asp Ile Trp Gly Gln 100 105 110Gly Thr Met Val Thr Val Ser Ser 115
12021110PRTArtificial SequenceSynthetic Polypeptide 21Gln Tyr Glu
Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln1 5 10 15Arg Val
Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser His 20 25 30Tyr
Val Phe Trp Tyr Gln Gln Leu Pro Gly Ala Ala Pro Lys Leu Leu 35 40
45Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu
Arg65 70 75 80Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Thr Trp Asp
Asn Ser Leu 85 90 95Ser Ala Trp Val Phe Gly Gly Gly Thr Lys Leu Thr
Val Leu 100 105 11022117PRTArtificial SequenceSynthetic Polypeptide
22Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Lys
Tyr 20 25 30Thr Met Trp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ser Val Ile Ser Ser Ser Gly Gly Lys Thr Tyr Tyr Ala
Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Thr Ala Asn Arg Ala Phe Asp
Ile Trp Gly Gln Gly Thr Met 100 105 110Val Thr Val Ser Ser
11523108PRTArtificial SequenceSynthetic Polypeptide 23Gln Asp Ile
Gln Met Thr Gln Ser Pro Ala Ala Leu Ser Val Ser Pro1 5 10 15Gly Glu
Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser 20 25 30Asp
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40
45Ile His Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser
50 55 60Gly Ser Gly Ser Gly Arg Glu Phe Thr Leu Thr Ile Ser Ser Leu
Gln65 70 75 80Ser Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asn
Asp Trp Pro 85 90 95Pro Leu Phe Gly Pro Gly Thr Lys Val Asn Ile Lys
100 10524125PRTArtificial SequenceSynthetic Polypeptide 24Glu Val
Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr 20 25
30Tyr Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ser Gly Ile Val Pro Ser Gly Gly Gln Thr Gly Tyr Ala Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Arg Thr Arg Arg Gly Trp Phe Gly Glu Asp
Tyr Tyr Tyr Tyr Met 100 105 110Asp Val Trp Gly Lys Gly Thr Leu Val
Thr Val Ser Ser 115 120 12525111PRTArtificial SequenceSynthetic
Polypeptide 25Gln Asp Ile Gln Met Thr Gln Ser Pro Gly Thr Leu Ser
Leu Ser Pro1 5 10 15Gly Glu Arg Ala Thr Val Ser Cys Arg Ala Ser Gln
Ser Val Gly Ser 20 25 30Thr Tyr Leu Ala Trp Tyr Gln His Lys Pro Gly
Gln Ala Pro Arg Leu 35 40 45Leu Ile Tyr Gly Ala Ser Ser Arg Ala Thr
Gly Ile Pro Asp Arg Phe 50 55 60Ser Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Ser Leu65 70 75 80Glu Pro Glu Asp Phe Ala Ile
Tyr Tyr Cys Gln His Phe His Thr Ser 85 90 95Pro Pro Gly Ile Thr Phe
Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105 11026118PRTArtificial
SequenceSynthetic Polypeptide 26Glu Val Gln Leu Leu Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Met Tyr 20 25 30Lys Met Ser Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Ile Ser Pro Ser
Gly Gly Arg Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg
Gly Thr Arg Thr Ser Gly Leu Asp Tyr Trp Gly Gln Gly Thr 100 105
110Leu Val Thr Val Ser Ser 11527110PRTArtificial SequenceSynthetic
Polypeptide 27Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser
Pro Gly Gln1 5 10 15Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp
Val Gly Gly Tyr 20 25 30Lys Tyr Val Ser Trp Tyr Gln Gln His Pro Gly
Lys Ala Pro Lys Leu 35 40 45Val Ile Tyr Glu Val Ser Asn Arg Pro Ser
Gly Val Ser Asn Arg Phe 50 55 60Ser Gly Ser Lys Ser Gly Asn Thr Ala
Ser Leu Thr Ile Ser Gly Leu65 70 75 80Gln Ala Glu Asp Glu Ala Asp
Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser 85 90 95Thr Thr Val Val Phe Gly
Gly Gly Thr Lys Leu Thr Val Leu 100 105 11028119PRTArtificial
SequenceSynthetic Polypeptide 28Glu Val Gln Leu Leu Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30Gly Met Arg Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Ile Ser Pro Ser
Gly Gly Lys Thr Asn Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg
Gly Arg Pro Asp Tyr Tyr Ala Met Asp Val Trp Gly Gln Gly 100 105
110Thr Thr Val Thr Val Ser Ser 11529110PRTArtificial
SequenceSynthetic Polypeptide 29Gln Ser Ala Leu Thr Gln Pro Pro Ser
Ala Ser Gly Ala Pro Gly Gln1 5 10 15Arg Val Thr Ile Ser Cys Ser Gly
Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30Thr Val Asn Trp Tyr Gln Lys
Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45Ile Tyr Tyr Asn Asp Arg
Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60Gly Ser Lys Ser Gly
Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu Gln65 70 75 80Ala Glu Asp
Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu 85 90 95Ser Gly
Pro Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105
11030126PRTArtificial SequenceSynthetic Polypeptide 30Glu Val Gln
Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30Pro
Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ser Gly Ile Ser Pro Ser Gly Gly Lys Thr Ala Tyr Ala Asp Ser Val
50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95Ala Arg Gly Gln Gly Arg Ala Val Arg Gly Lys Leu
Tyr Tyr Tyr Gly 100 105 110Met Asp Val Trp Gly Gln Gly Thr Thr Val
Thr Val Ser Ser 115 120 12531110PRTArtificial SequenceSynthetic
Polypeptide 31Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gln Thr
Pro Gly Gln1 5 10 15Thr Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn
Ile Gly Thr Asn 20 25 30Asn Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr
Ala Pro Lys Leu Leu 35 40 45Ile Ser Ser His His Arg Arg Pro Ser Gly
Val Pro Asp Arg Phe Ser 50 55 60Ala Ser Lys Ser Gly Thr Ser Ala Ser
Leu Ala Ile Ser Gly Leu Gln65 70 75 80Ser Glu Asp Glu Ala Asp Tyr
Tyr Cys Ala Ala Trp Asp Asp Ser Leu 85 90 95Asn Gly Pro Val Phe Gly
Gly Gly Thr Lys Leu Thr Val Leu 100 105 11032117PRTArtificial
SequenceSynthetic Polypeptide 32Glu Val Gln Leu Leu Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Met Tyr 20 25 30His Met Asn Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ser Ile Tyr Ser Ser
Gly Gly Ser Thr Arg Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg
Gly Val Arg Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr 100 105
110Val Thr Val Ser Ser 11533108PRTArtificial SequenceSynthetic
Polypeptide 33Gln Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser
Ala Ser Val1 5 10 15Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
Gly Ile Ser Ser 20 25 30Trp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys
Ala Pro Lys Leu Leu 35 40 45Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly
Val Pro Ser Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Ser Ser Leu Gln65 70 75 80Pro Glu Asp Phe Ala Thr Tyr
Tyr Cys Gln Gln Ala Asn Ser Phe Pro 85 90 95Ile Thr Phe Gly Gln Gly
Thr Arg Leu Glu Ile Lys 100 10534122PRTArtificial SequenceSynthetic
Polypeptide 34Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Thr Phe Ser Met Tyr 20 25 30Asp Met His Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45Ser Ser Ile Ser Ser Ser Gly Gly Tyr
Thr Gln Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95Ala Arg Asp Arg Gly
Leu Ile Ala Ala Ala Gly Gly Phe Asp Pro Trp 100 105 110Gly Gln Gly
Thr Leu Val Thr Val Ser Ser 115 12035108PRTArtificial
SequenceSynthetic Polypeptide 35Gln Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val1 5 10 15Gly Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Gln Ser Ile Gly Ile 20 25 30Tyr Leu Asn Trp Tyr Gln Gln
Lys Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45Ile Tyr Ala Ala Ser Ser
Leu Gln Ser Gly Val Pro Ser Arg Phe Thr 50 55 60Gly Ser Gly Ser Gly
Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln65 70 75 80Pro Asp Asp
Phe Ala Thr Tyr Tyr Cys Gln Arg Thr Tyr Gly Arg Pro 85 90 95Leu Thr
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 10536121PRTArtificial
SequenceSynthetic Polypeptide 36Glu Val Gln Leu Leu Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Lys Tyr 20 25 30Glu Met Met Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ser Ile Ser Pro Ser
Gly Gly Tyr Thr Met Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg
His Arg Ser Lys Trp Asn Asp Ala Pro Phe Asp Ser Trp Gly 100 105
110Gln Gly Thr Leu Val Thr Val Ser Ser 115 12037109PRTArtificial
SequenceSynthetic Polypeptide 37Gln Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val1 5 10 15Gly Asp Arg Val Ala Ile Thr Cys
Arg Ala Ser Gln Ser Ile Asp Thr 20 25 30Tyr Leu Asn Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45Ile Tyr Ala Ala Ser Lys
Leu Glu Asp Gly Val Pro Ser Arg Phe Ser 50 55 60Gly Ser Gly Thr Gly
Thr Asp Phe Thr Leu Thr Ile Arg Ser Leu Gln65 70 75 80Pro Glu Asp
Phe Ala Ser Tyr Phe Cys Gln Gln Ser Tyr Ser Ser Pro 85 90 95Gly Ile
Thr Phe Gly Pro Gly Thr Lys Val Glu Ile Lys 100
10538123PRTArtificial SequenceSynthetic Polypeptide 38Glu Val Gln
Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30Gln
Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ser Ser Ile Tyr Ser Ser Gly Gly Arg Thr Phe Tyr Ala Asp Ser Val
50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95Ala Thr Arg Gly Ser Trp Tyr Val Gly Gly Asn Glu
Tyr Phe Gln His 100 105 110Trp Gly Gln Gly Thr Leu Val Thr Val Ser
Ser 115 12039106PRTArtificial SequenceSynthetic Polypeptide 39Gln
Ser Val Leu Thr Gln Ser Pro Ser Leu Ser Leu Ser Pro Gly Gln1 5 10
15Thr Ala Ser Ile Pro Cys Ser Gly Asp Thr Leu Gly Asn Lys Phe Val
20 25 30Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Val Leu Val Ile
Tyr 35 40 45Gln Asp Thr Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser
Gly Ser 50 55 60Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Thr Gly Thr
Gln Ala Met65 70 75 80Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp
Ser Asn Ser Tyr Ala 85 90 95Phe Gly Pro Gly Thr Lys Val Thr Val Leu
100 10540120PRTArtificial SequenceSynthetic Polypeptide 40Glu Val
Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Phe Tyr 20 25
30Met Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ser Ser Ile Ser Ser Ser Gly Gly Phe Thr Arg Tyr Ala Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Arg Val Arg Gly Leu Ala Val Ala Ala Pro
Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115
12041110PRTArtificial SequenceSynthetic Polypeptide 41Gln Ser Glu
Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln1 5 10 15Ser Ile
Thr Ile Ser Cys Ile Gly Thr Ser Ser Asp Ile Gly Thr Tyr 20 25 30Asn
Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40
45Met Ile Tyr Asp Val Asn Thr Arg Pro Ser Gly Val Ser Asp Arg Phe
50 55 60Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly
Leu65 70 75 80Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr
Thr Thr Ser 85 90 95Val Thr Trp Val Phe Gly Gly Gly Thr Thr Leu Thr
Val Leu 100 105 11042115PRTArtificial SequenceSynthetic Polypeptide
42Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly
Tyr 20 25 30Asn Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ser Arg Ile Ser Pro Ser Gly Gly Trp Thr Ser Tyr Ala
Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Thr Arg Gly Gln Trp Met Asp Trp Trp
Gly Gln Gly Thr Met Val Thr 100 105 110Val Ser Ser
11543108PRTArtificial SequenceSynthetic Polypeptide 43Gln Asp Ile
Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val1 5 10 15Gly Asp
Arg Val Ile Ile Thr Cys Arg Ala Ser Gln Asn Ile Thr Gly 20 25 30Tyr
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Asn Leu Leu 35 40
45Ile Tyr Asp Ala Ser Arg Met Asn Thr Gly Val Pro Ser Arg Phe Arg
50 55 60Gly Ser Gly Ser Gly Thr Asp Tyr Ile Leu Thr Ile Tyr Lys Leu
Glu65 70 75 80Pro Glu Asp Ile Gly Thr Tyr Phe Cys Gln His Thr Asp
Asp Phe Ser 85 90 95Val Thr Phe Gly Gly Gly Thr Lys Val Asp Leu Lys
100 10544116PRTArtificial SequenceSynthetic Polypeptide 44Glu Val
Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe His Tyr Arg 20 25
30Met Met Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser
35 40 45Tyr Ile Ser Ser Ser Gly Gly Tyr Thr Ala Tyr Ala Asp Ser Val
Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr Leu65 70 75 80Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys Ala 85 90 95Ala Lys Arg Asn Arg Ala Phe Asp Ile Trp Gly
Gln Gly Thr Met Val 100 105 110Thr Val Ser Ser
11545114PRTArtificial SequenceSynthetic Polypeptide 45Gln Asp Ile
Gln Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu1 5 10 15Gly Glu
Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr 20 25 30Ser
Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly 35 40
45Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly
50 55 60Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu65 70 75 80Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr
Tyr Cys Gln 85 90 95Gln Tyr Tyr Ser Thr Pro Leu Gly Phe Gly Gln Gly
Thr Lys Leu Glu 100 105 110Ile Lys46120PRTArtificial
SequenceSynthetic Polypeptide 46Glu Val Gln Leu Leu Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Arg Tyr 20 25 30Gln Met Thr Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ser Ile Gly Ser Ser
Gly Gly Phe Thr Asn Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg
Leu Pro Ala Asn Phe Tyr Tyr Tyr Met Asp Val Trp Gly Lys 100 105
110Gly Thr Thr Val Thr Val Ser Ser 115 12047108PRTArtificial
SequenceSynthetic Polypeptide 47Gln Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val1 5 10 15Gly Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Gln Asn Ile Tyr Ser 20 25 30Phe Leu Asn Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45Ile Tyr Ala Thr Ser Ser
Leu Gln Ser Gly Val Pro Ser Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly
Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln65 70 75 80Pro Glu Asp
Phe Ala Ser Tyr Tyr Cys Gln Gln Asn Tyr Asn Ile Pro 85 90 95Trp Thr
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 10548123PRTArtificial
SequenceSynthetic Polypeptide 48Glu Val Gln Leu Leu Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Trp Tyr 20 25 30Met Met Lys Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ser Ile Val Pro Ser
Gly Gly Trp Thr Thr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Thr
Glu Gly Asn Leu Trp Phe Gly Glu Gly Arg Ala Phe Asp Ile 100 105
110Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 115
12049109PRTArtificial SequenceSynthetic Polypeptide 49Gln Asp Ile
Gln Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro1 5 10 15Gly Glu
Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser 20 25 30Ser
Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu 35 40
45Leu Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe
50 55 60Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg
Leu65 70 75 80Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg
Ser Asn Trp 85 90 95Pro Pro Ser Phe Gly Gln Gly Thr Arg Leu Asp Ile
Lys 100 10550126PRTArtificial SequenceSynthetic Polypeptide 50Glu
Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Lys Tyr
20 25 30Asp Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45Ser Arg Ile Ser Ser Ser Gly Gly Lys Thr Glu Tyr Ala Asp
Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Arg Glu Tyr Arg Tyr Cys Thr Ala Asn
Thr Cys Ser Leu Tyr Gly 100 105 110Met Asp Val Trp Gly Arg Gly Thr
Thr Val Thr Val Ser Ser 115 120 12551108PRTArtificial
SequenceSynthetic Polypeptide 51Gln Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val1 5 10 15Gly Asp Arg Val Ala Ile Thr Cys
Arg Thr Ser Gln Gly Val Arg Ser 20 25 30Asp Phe Ala Trp Tyr Gln Gln
Thr Pro Gly Lys Ala Pro Arg Arg Leu 35 40 45Ile Tyr Ala Ala Phe Ile
Leu Asp Asn Gly Val Pro Ser Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly
Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln65 70 75 80Pro Glu Asp
Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro 85 90 95Leu Thr
Phe Gly Gly Gly Thr Lys Val Glu Met Lys 100 10552120PRTArtificial
SequenceSynthetic Polypeptide 52Glu Val Gln Leu Leu Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30Trp Met His Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Ile Ser Pro Ser
Gly Gly Gly Thr Gly Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg
Glu Ser Arg Gly Ser Gly Ser His Glu Asp Tyr Trp Gly Gln 100 105
110Gly Thr Leu Val Thr Val Ser Ser 115 12053109PRTArtificial
SequenceSynthetic Polypeptide 53Gln Asp Ile Gln Met Thr Gln Ser Pro
Ala Thr Leu Ser Leu Ser Pro1 5 10 15Gly Glu Arg Ala Thr Leu Ser Cys
Arg Ala Ser Gln Ser Val Ser Ser 20 25 30Tyr Leu Ala Trp Tyr Gln Gln
Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45Ile Tyr Gly Ala Ser Asn
Arg Gly Thr Gly Ile Pro Ala Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly
Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln65 70 75 80Ser Glu Asp
Phe Ala Val Tyr Phe Cys Gln Gln Tyr Lys Asn Trp Pro 85 90 95Asn Leu
Thr Phe Gly Gly Gly Thr Lys Val Asp Ile Lys 100
10554122PRTArtificial SequenceSynthetic Polypeptide 54Glu Val Gln
Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser His Tyr 20 25 30Pro
Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ser Gly Ile Val Ser Ser Gly Gly Arg Thr Val Tyr Ala Asp Ser Val
50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp Pro Tyr Asp Phe Trp Ser Glu
Gly Ala Phe Asp Ile Trp 100 105 110Gly Gln Gly Thr Met Val Thr Val
Ser Ser 115 12055111PRTArtificial SequenceSynthetic Polypeptide
55Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln1
5 10 15Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn
Asn 20 25 30Phe Val Tyr Trp Tyr His Gln Val Pro Gly Thr Ala Pro Lys
Leu Leu 35 40 45Ile Tyr Lys Asn Asn Gln Arg Pro Ser Gly Val Pro Asp
Arg Phe Ser 50 55 60Gly Ser Lys Ser Ala Ala Ser Ala Ser Leu Ala Ile
Ser Gly Leu Arg65 70 75 80Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala
Ala Trp Asp Asn Ser Leu 85 90 95Ser Gly Phe Tyr Val Phe Gly Ala Gly
Thr Lys Val Thr Val Leu 100 105 11056120PRTArtificial
SequenceSynthetic Polypeptide 56Glu Val Gln Leu Leu Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Trp Tyr 20 25 30Gly Met His Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Arg Ile Gly Pro Ser
Gly Gly Pro Thr Ser Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg
Gly Tyr Tyr Gly Thr Gly Arg Tyr Phe Gln His Trp Gly Gln 100 105
110Gly Thr Leu Val Thr Val Ser Ser 115 12057109PRTArtificial
SequenceSynthetic Polypeptide 57Gln Asp Ile Gln Met Thr Gln Ser Pro
Asp Ser Leu Ser Leu Ser Pro1 5 10 15Gly Asp Arg Ala Thr Leu Ser Cys
Arg Ala Ser Gln Ser Val Gly Ser 20 25 30Asp Tyr Leu Ala Trp Tyr Gln
Gln Lys Pro Gly Gln Ala Pro Arg Leu 35 40 45Leu Ile Tyr Asp Ala Ser
Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe 50 55 60Ser Gly Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu65 70 75 80Glu Pro Glu
Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp 85 90 95Pro Pro
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100
10558116PRTArtificial SequenceSynthetic Polypeptide 58Glu Val Gln
Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ala Tyr 20 25 30Ala
Met Arg Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ser Tyr Ile Ser Ser Ser Gly Gly Glu Thr Met Tyr Ala Asp Ser Val
50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95Ala Asn Gly Tyr Gly Arg Ile Asp Tyr Trp Gly Gln
Gly Thr Leu Val 100 105 110Thr Val Ser Ser 11559110PRTArtificial
SequenceSynthetic Polypeptide 59Gln Ser Val Leu Thr Gln Pro Ala Ser
Val Ser Gly Ser Pro Gly Gln1 5 10 15Ser Ile Thr Ile Ser Cys Thr Gly
Thr Ser Ser Asp Ile Gly Gly Tyr 20 25 30Asn Tyr Val Ser Trp Tyr Gln
Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45Met Ile Tyr Glu Val Ser
Asn Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55 60Ser Gly Ser Lys Ser
Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu65 70 75 80Gln Ala Glu
Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Gly 85 90 95Ser Thr
Arg Val Phe Gly Thr Gly Thr Arg Val Thr Val Leu 100 105
11060121PRTArtificial SequenceSynthetic Polypeptide 60Glu Val Gln
Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ala Tyr 20 25 30Val
Met Arg Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ser Ser Ile Gly Ser Ser Gly Gly Pro Thr Tyr Tyr Ala Asp Ser Val
50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95Ala Arg Arg Gly Gly Ser Gly Ser Ser His Ala Phe
Asp Ile Trp Gly 100 105 110Gln Gly Thr Met Val Thr Val Ser Ser 115
12061108PRTArtificial SequenceSynthetic Polypeptide 61Gln Asp Ile
Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val1 5 10 15Gly Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser 20 25 30Tyr
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 35 40
45Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser
50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Gln65 70 75 80Pro Glu Asp Ser Gly Thr Tyr Tyr Cys Gln Gln Tyr Asn
Ser Phe Pro 85 90 95Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 10562122PRTArtificial SequenceSynthetic Polypeptide 62Glu Val
Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr Tyr 20 25
30Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ser Val Ile Ser Pro Ser Gly Gly Leu Thr Val Tyr Ala Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Met Tyr Tyr Cys 85 90 95Ala Thr Gly Phe Ala Val Gln His Gly Gly Gly
Ala Phe Asp Ile Trp 100 105 110Gly Gln Gly Thr Met Val Thr Val Ser
Ser 115 12063108PRTArtificial SequenceSynthetic Polypeptide 63Gln
Asp Ile Gln Met Thr Gln Ser Pro Ala Thr Leu Ser Met Ser Pro1 5 10
15Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Thr Thr
20 25 30Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu
Leu 35 40 45Ile Tyr Asp Ala Ser Ile Arg Ala Thr Gly Val Pro Ala Arg
Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Arg Leu Glu65 70 75 80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln
Arg Thr Ile Trp Pro 85 90 95Leu Thr Phe Gly Gly Gly Thr Lys Val Glu
Ile Lys 100 10564119PRTArtificial SequenceSynthetic Polypeptide
64Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro
Tyr 20 25 30Glu Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ser Ser Ile Val Pro Ser Gly Gly Trp Thr Val Tyr Ala
Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Ser Pro Ser Gly Arg Gly Leu Ala
Phe Asp Ile Trp Gly Gln Gly 100 105 110Thr Met Val Thr Val Ser Ser
11565109PRTArtificial SequenceSynthetic Polypeptide 65Gln Asp Ile
Gln Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro1 5 10 15Gly Glu
Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Ser 20 25 30Ser
Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu 35 40
45Leu Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Val Pro Asp Arg Phe
50 55 60Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser
Leu65 70 75 80Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Gln
Lys Ser Tyr 85 90 95Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys 100 10566119PRTArtificial SequenceSynthetic Polypeptide 66Glu
Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Lys Tyr
20 25 30Phe Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45Ser Trp Ile Ser Ser Ser Gly Gly Tyr Thr Asn Tyr Ala Asp
Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ala Tyr Tyr Tyr Asp Ala Phe
Asp Ile Trp Gly Gln Gly 100 105 110Thr Met Val Thr Val Ser Ser
11567108PRTArtificial SequenceSynthetic Polypeptide 67Gln Asp Ile
Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val1 5 10 15Gly Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ala Ile 20 25 30Phe
Leu Asn Trp Tyr Gln Gln Thr Pro Gly Lys Pro Pro Lys Leu Leu 35 40
45Ile Tyr Gly Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser
50 55 60Gly Ser Gly Ser Gly Ala Asp Phe Thr Leu Thr Ile Ser Asn Leu
Gln65 70 75 80Leu Glu Asp Phe Thr Thr Tyr Tyr Cys Gln Gln Ser Tyr
Ser Thr Leu 85 90 95Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 10568117PRTArtificial SequenceSynthetic Polypeptide 68Glu Val
Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr 20 25
30Ser Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ser Val Ile Ser Ser Ser Gly Gly Met Thr Tyr Tyr Ala Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Met Tyr Tyr Cys 85 90 95Ala Arg Asp Tyr Tyr Gly Asn Met Asp Val Trp
Gly Lys Gly Thr Thr 100 105 110Val Thr Val Ser Ser
11569108PRTArtificial SequenceSynthetic Polypeptide 69Gln Asp Ile
Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Thr Ser Val1 5 10 15Gly Asp
Arg Val Thr Ile Thr Cys Arg Thr Ser Gln Asp Ile Ser Gly 20 25 30Ala
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Arg Leu Leu 35 40
45Ile Phe Gly Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser
50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Gln65 70 75 80Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn
Lys Tyr Pro 85 90 95Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 10570116PRTArtificial SequenceSynthetic Polypeptide 70Glu Val
Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Trp Tyr 20 25
30Thr Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ser Tyr Ile Tyr Pro Ser Gly Gly Tyr Thr Met Tyr Ala Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Asn Pro Tyr Ser Ser Gly Gly Tyr Trp Gly
Gln Gly Thr Leu Val 100 105 110Thr Val Ser Ser
11571113PRTArtificial SequenceSynthetic Polypeptide 71Gln Asp Ile
Gln Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro1 5 10 15Gly Glu
Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Asp 20 25 30Ser
Asn Gly Tyr Asn Tyr Leu Asp Trp Phe Leu Gln Lys Pro Gly Gln 35 40
45Ser Pro Gln Leu Leu Ile Tyr Leu Gly Phe Asn Arg Ala Ser Gly Val
50 55 60Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
Lys65 70 75 80Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr
Cys Met Gln 85 90 95Ala Leu Gln Thr Pro Tyr Thr Phe Gly Gln Gly Thr
Lys Leu Glu Ile 100 105 110Thr72120PRTArtificial SequenceSynthetic
Polypeptide 72Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Ala Tyr 20 25 30Leu Met Thr Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu Glu Trp Val 35 40 45Ser Gly Ile Ser Pro Ser Gly Gly Ile Thr
Lys Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp Ile Pro Asn
Trp Ile Tyr Gly Met Asp Val Trp Gly Gln 100 105 110Gly Thr Thr Val
Thr Val Ser Ser 115 12073105PRTArtificial SequenceSynthetic
Polypeptide 73Gln Ser Ala Leu Thr Gln Pro Pro Ser Val Ser Val Ser
Pro Gly Gln1 5 10 15Thr Ala Ser Ile Thr Cys Ser Gly Asp Lys Leu Gly
Asn Lys Tyr Ala 20 25 30Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro
Val Leu Val Ile Tyr 35 40 45Gln Asp Arg Arg Arg Pro Ser Gly Ile Pro
Glu Arg Phe Ser Gly Ser 50 55 60Asn Ser Gly Asn Thr Ala Thr Leu Thr
Ile Ser Gly Thr Gln Ala Met65 70 75 80Asp Glu Ala Asp Tyr Tyr Cys
Gln Ala Trp Asp Ser Gly Val Val Phe 85 90 95Gly Gly Gly Thr Lys Leu
Thr Val Leu 100 10574124PRTArtificial SequenceSynthetic Polypeptide
74Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn
Tyr 20 25 30Leu Met Leu Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ser Gly Ile Ser Pro Ser Gly Gly Gly Thr Ala Tyr Ala
Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Met Ala Val Tyr Tyr Cys 85 90 95Ala Lys Val Ala Tyr Ser Gly Ser Tyr
Tyr Tyr Tyr Tyr Tyr Met Asp 100 105 110Val Trp Gly Lys Gly Thr Thr
Val Thr Val Ser Ser 115 12075109PRTArtificial SequenceSynthetic
Polypeptide 75Gln Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser
Ala Ser
Val1 5 10 15Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile
Ser Ser 20 25 30Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Leu Leu 35 40 45Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro
Ser Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln65 70 75 80Pro Glu Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln Ser Tyr Ser Thr His 85 90 95Ser Ile Thr Phe Gly Gln Gly Thr
Arg Leu Glu Ile Lys 100 10576137PRTArtificial SequenceSynthetic
Polypeptide 76Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Gln Tyr 20 25 30Ile Met Gly Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu Glu Trp Val 35 40 45Ser Ser Ile Gly Ser Ser Gly Val Thr Val
Tyr Ala Asp Ser Val Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr Leu Tyr Leu65 70 75 80Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Arg Gly Gly Gly Val Thr
Val Leu His Ala Phe Asp Ile Trp Gly Gln 100 105 110Gly Thr Met Val
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125Phe Pro
Leu Ala Pro Ser Ser Lys Ser 130 13577118PRTArtificial
SequenceSynthetic Polypeptide 77Gln Ser Ala Leu Thr Gln Pro Ala Ser
Val Ser Gly Ser Pro Gly Gln1 5 10 15Ser Ile Thr Ile Ser Cys Thr Gly
Thr Ser Ser Asp Val Gly Gly Tyr 20 25 30Asn Tyr Val Ser Trp Tyr Gln
Gln His Pro Gly Lys Val Pro Lys Leu 35 40 45Ile Ile Tyr Glu Gly Asn
Lys Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60Ser Gly Ser Lys Ala
Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu65 70 75 80Gln Ala Glu
Asp Glu Ala Asp Tyr Tyr Cys Thr Ala Tyr Gly Gly His 85 90 95Ser Arg
Phe Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly 100 105
110Gln Pro Lys Ala Asn Pro 11578304PRTHomo sapiens 78Met Ile Tyr
Thr Met Lys Lys Val His Ala Leu Trp Ala Ser Val Cys1 5 10 15Leu Leu
Leu Asn Leu Ala Pro Ala Pro Leu Asn Ala Asp Ser Glu Glu 20 25 30Asp
Glu Glu His Thr Ile Ile Thr Asp Thr Glu Leu Pro Pro Leu Lys 35 40
45Leu Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly Pro Cys Lys
50 55 60Ala Ile Met Lys Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys
Glu65 70 75 80Glu Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg
Phe Glu Ser 85 90 95Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp Asn
Ala Asn Arg Ile 100 105 110Ile Lys Thr Thr Leu Gln Gln Glu Lys Pro
Asp Phe Cys Phe Leu Glu 115 120 125Glu Asp Pro Gly Ile Cys Arg Gly
Tyr Ile Thr Arg Tyr Phe Tyr Asn 130 135 140Asn Gln Thr Lys Gln Cys
Glu Arg Phe Lys Tyr Gly Gly Cys Leu Gly145 150 155 160Asn Met Asn
Asn Phe Glu Thr Leu Glu Glu Cys Lys Asn Ile Cys Glu 165 170 175Asp
Gly Pro Asn Gly Phe Gln Val Asp Asn Tyr Gly Thr Gln Leu Asn 180 185
190Ala Val Asn Asn Ser Leu Thr Pro Gln Ser Thr Lys Val Pro Ser Leu
195 200 205Phe Glu Phe His Gly Pro Ser Trp Cys Leu Thr Pro Ala Asp
Arg Gly 210 215 220Leu Cys Arg Ala Asn Glu Asn Arg Phe Tyr Tyr Asn
Ser Val Ile Gly225 230 235 240Lys Cys Arg Pro Phe Lys Tyr Ser Gly
Cys Gly Gly Asn Glu Asn Asn 245 250 255Phe Thr Ser Lys Gln Glu Cys
Leu Arg Ala Cys Lys Lys Gly Phe Ile 260 265 270Gln Arg Ile Ser Lys
Gly Gly Leu Ile Lys Thr Lys Arg Lys Arg Lys 275 280 285Lys Gln Arg
Val Lys Ile Ala Tyr Glu Glu Ile Phe Val Lys Asn Met 290 295
3007958PRTHomo sapiens 79Arg Pro Asp Phe Cys Leu Glu Pro Pro Tyr
Thr Gly Pro Cys Lys Ala1 5 10 15Arg Ile Ile Arg Tyr Phe Tyr Asn Ala
Lys Ala Gly Leu Cys Gln Thr 20 25 30Phe Val Tyr Gly Gly Cys Arg Ala
Lys Arg Asn Asn Phe Lys Ser Ala 35 40 45Glu Asp Cys Met Arg Thr Cys
Gly Gly Ala 50 558060PRTArtificial SequenceSynthetic Polypeptide
80Glu Ala Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly Pro Cys1
5 10 15Arg Ala Ala His Pro Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln
Cys 20 25 30Glu Glu Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg
Phe Glu 35 40 45Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50
55 6081122PRTArtificial SequenceSynthetic Polypeptide 81Glu Val Gln
Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser His Tyr 20 25 30Ile
Met Met Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ser Gly Ile Tyr Ser Ser Gly Gly Ile Thr Val Tyr Ala Asp Ser Val
50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95Ala Tyr Arg Arg Ile Gly Val Pro Arg Arg Asp Glu
Phe Asp Ile Trp 100 105 110Gly Gln Gly Thr Met Val Thr Val Ser Ser
115 12082105PRTArtificial SequenceSynthetic Polypeptide 82Asp Ile
Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly1 5 10 15Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp 20 25
30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45Tyr Lys Ala Ser Thr Leu Glu Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu
Gln Pro65 70 75 80Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn
Thr Tyr Trp Thr 85 90 95Phe Gly Gln Gly Thr Lys Val Glu Ile 100
1058320PRTArtificial SequenceSynthetic Polypeptide 83His Lys His
Gly His Gly His Gly Lys His Lys Asn Lys Gly Lys Lys1 5 10 15Asn Gly
Lys His 20
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References